Automated cell processing methods, modules, instruments, and systems comprising flow-through electroporation devices

ABSTRACT

In an illustrative embodiment, automated multi-module cell editing instruments comprising one or more flow-through electroporation devices or modules are provided to automate genome editing in live cells.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/426,310, entitled “Automated Cell Processing Methods, Modules,Instruments, and Systems Comprising Flow-through ElectroporationDevices,” filed May 30, 2019, which is a continuation of U.S. patentapplication Ser. No. 16/147,865, entitled “Automated Cell ProcessingMethods, Modules, Instruments, and Systems Comprising Flow-throughElectroporation Devices,” filed Sep. 30, 2018, which claims priority toU.S. Patent Application Ser. No. 62/566,374, entitled “ElectroporationDevice,” filed Sep. 30, 2017; U.S. Patent Application Ser. No.62/566,375, entitled “Electroporation Device,” filed Sep. 30, 2017; U.S.Patent Application Ser. No. 62/566,688, entitled “Introduction ofExogenous Materials into Cells,” filed Oct. 2, 2017; U.S. PatentApplication Ser. No. 62/567,697, entitled “Automated Nucleic AcidAssembly and Introduction of Nucleic Acids into Cells,” filed Oct. 3,2017; U.S. Patent Application Ser. No. 62/620,370, entitled “AutomatedFiltration and Manipulation of Viable Cells,” filed Jan. 22, 2018; U.S.Patent Application Ser. No. 62/649,731, entitled “Automated Control ofCell Growth Rates for Induction and Transformation,” filed Mar. 29,2018; U.S. Patent Application Ser. No. 62/671,385, entitled “AutomatedControl of Cell Growth Rates for Induction and Transformation,” filedMay 14, 2018; U.S. Patent Application Ser. No. 62/648,130, entitled“Genomic Editing in Automated Systems,” filed Mar. 26, 2018; U.S. PatentApplication Ser. No. 62/657,651, entitled “Combination Reagent Cartridgeand Electroporation Device,” filed Apr. 13, 2018; U.S. PatentApplication Ser. No. 62/657,654, entitled “Automated Cell ProcessingSystems Comprising Cartridges,” filed Apr. 13, 2018; and U.S. PatentApplication Ser. No. 62/689,068, entitled “Nucleic Acid PurificationProtocol for Use in Automated Cell Processing Systems,” filed Jun. 23,2018. All above identified applications are hereby incorporated byreference in their entireties for all purposes.

BACKGROUND

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

Genome editing with engineered nucleases is a method in which changes tonucleic acids are made in the genome of a living organism. Certainnucleases create site-specific double-strand breaks at target regions inthe genome, which can be repaired by nonhomologous end-joining orhomologous recombination, resulting in targeted edits. These methods,however, have not been compatible with automation due to lowefficiencies and challenges with cell transformation, growthmeasurement, and cell selection. Moreover, traditional benchtop devicesdo not necessarily scale and integrate well into an automated, modularinstrument or system. Methods and instruments to create edited cellpopulations thus remain cumbersome—including methods and instruments forautomated cell transformation—and the challenges of introducing multiplerounds of edits using recursive techniques has limited the nature andcomplexity of the cell populations that can be created.

There is thus a need for automated instruments, systems and methods forintroducing assembled nucleic acids and other biological molecules intoliving cells in an automated fashion where the edited cells may be usedfor further experimentation outside of the automated instrument.

SUMMARY OF ILLUSTRATIVE EMBODIMENTS

In certain embodiments, automated modules, instruments, systems andmethods are used for nuclease-directed genome editing of one or moretarget genomic regions in multiple cells, the methods being performed inautomated multi-module cell editing instruments. These methods can beused to generate libraries of living cells of interest with desiredgenomic changes. The automated methods carried out using the automatedmulti-module cell editing instruments described herein may employ avariety of nuclease-directed genome editing techniques, and can be usedwith or without use of one or more selectable markers.

The present disclosure thus provides, in selected embodiments, modules,instruments, and systems for automated multi-module cell editing,including nuclease-directed genome editing. In particular, theinstruments and systems comprise a flow-through electroporation (FTEP)device for transforming the cells to be edited. Other specificembodiments of the automated multi-module cell editing instruments ofthe disclosure are designed for recursive genome editing, e.g.,sequentially introducing multiple edits into genomes inside one or morecells of a cell population through two or more editing operations.

Thus, provided herein are embodiments of an automated multi-module cellediting instrument comprising: a housing configured to contain all orsome of the modules; a receptacle configured to receive cells; one ormore receptacles configured to receive nucleic acids; a transformationmodule configured to introduce the nucleic acids into the cells whereinthe transformation module comprises one or more FTEP devices; a recoverymodule configured to allow the cells to recover after celltransformation in the transformation module; an editing moduleconfigured to allow the nucleic acids transformed into the cells to editnucleic acids in the cells; and a processor configured to operate theautomated multi-module cell editing instrument based on user inputand/or selection of an appropriate controller script.

In some embodiments, the automated multi-module cell editing instrumentscomprise a flow-through electroporation (FTEP) device for introducing anexogenous material into cells in a fluid, where the FTEP devicecomprises: one or more inlets and inlet channels for introducing a fluidcomprising cells and exogenous material into the FTEP device; an outletand an outlet channel for removing a fluid comprising transformed cellsand exogenous material from the FTEP device; a flow channel intersectingand positioned between a first inlet channel and the outlet channel,wherein the flow channel decreases in width between the first inletchannel and the center of the flow channel and the outlet channel andthe center of the flow channel; and two or more electrodes positioned inthe flow channel between the intersection of the flow channel with thefirst inlet channel and the intersection of the flow channel with theoutlet channel, wherein the electrodes are in fluid communication withfluid in the flow channel but are not in the direct flow path of thecells in the flow channel, and wherein the electrodes apply one or moreelectric pulses to the cells in the fluid as they pass through the flowchannel, thereby introducing the exogenous material into the cells inthe fluid.

In some aspects of this embodiment, the two electrodes in the FTEPdevice are located from 0.5 mm to 10 mm apart, or from 1 mm to 8 mmapart, or from 3 mm and 7 mm apart, or from 4 mm to 6 mm apart.

In other embodiments, the automated multi-module cell editinginstruments comprise a flow-through electroporation (FTEP) device forintroducing an exogenous material into cells in a fluid, where the FTEPdevice comprises: at least one inlet and at least one inlet channel forintroducing a fluid comprising cells and exogenous material to the FTEPdevice; an outlet and an outlet channel for removing transformed cellsand exogenous material from the FTEP device; a flow channel positionedbetween a first inlet channel and the outlet channel, wherein the flowchannel intersects with the first inlet channel and the outlet channeland wherein a portion of the flow channel narrows between the inletchannel intersection and the outlet channel intersection; and anelectrode positioned on either side of the flow channel and in directcontact with the fluid in the flow channel, the electrodes defining thenarrowed portion of the flow channel, and wherein the electrodes applyone or more electric pulses to the cells in the fluid as they passthrough the flow channel, thereby introducing the exogenous materialinto the cells in the fluid.

In some aspects of this embodiment, the electrodes are positioned oneither side of the flow channel, are in direct contact with the fluid inthe flow channel and define the decrease in width of the flow channel.In some configurations of this aspect, the electrodes are between 10 μmto 5 mm apart, or between 25 μm to 2 mm apart.

In some aspects of these embodiments, the FTEP device is between 3 cm to15 cm in length, or between 4 cm to 12 cm in length, or from 4.5 cm to10 cm in length, or from 5 cm to 8 cm in length. In some aspects ofthese embodiments, this embodiment of the FTEP device is between 0.5 cmto 5 cm in width, or from 0.75 cm to 3 cm in width, or from 1 cm to 2.5cm in width, or from 1 cm to 1.5 cm in width. In some aspects of theseembodiments, the narrowest part of the channel width in the FTEP deviceis from 10 μM to 5 mm such that whatever cell type is being transformedwill not be physically contorted or “squeezed” by features of the FTEPdevice.

Also in some aspects of these embodiments, the flow rate in the FTEPranges from 0.1 ml to 5 ml per minute, or from 0.5 ml to 3 ml perminute, or from 1 ml to 2.5 ml per minute. In some aspects of theseembodiments the electrodes are configured to deliver 1-25 Kv/cm, or10-20 Kv/cm.

In some aspects of these embodiments, the FTEP device further comprisesone or more filters between the one or more inlet channels and theoutlet channel. In some aspects, there are two filters, one between theinlet channel and the narrowed portion of the flow channel, and onebetween the narrowed portion of the flow channel and the outlet channel.In some aspects of these embodiments, the filters are graduated in poresize with the larger pores proximal to the inlet chamber or outletchamber, and the small pores proximal to the narrowed portion of theflow channel. In some aspects, the small pores are the same size orlarger than the size of the narrowed portion of the flow channel. Insome aspects of these embodiments, the filter is formed separately fromthe body of the FTEP device and placed into the FTEP device as it isbeing assembled. Alternatively, in some aspects of these embodiments,the filter may be formed as part of and integral to the body of the FTEPdevice.

In some aspects of these embodiments, the FTEP device further comprisesa reservoir connected to the inlet for introducing the cells in fluidinto the FTEP device and a reservoir connected to the outlet forremoving transformed cells from the FTEP device, and in some aspects,the FTEP device comprises two inlets and two inlet channels and furthercomprises a reservoir connected to a second inlet for introducing theexogenous material into the FTEP device. In some aspects the FTEP devicecomprises a reservoir connected to the inlet for introducing both thecells in fluid and the exogenous material into the FTEP device and areservoir connected to the outlet for removing transformed cells fromthe FTEP device In some aspects of these embodiments, the reservoirscoupled to the inlet(s) and outlet range in volume from 100 μL to 10 ml,or from 0.5 ml to 7 ml, or from 1 ml to 5 ml.

In some aspects of these embodiments, the FTEP devices can provide acell transformation rate of 10³ to 10¹² cells per minute, or 10⁴ to 10¹⁰per minute, or 10⁵ to 10⁹ per minute, or 10⁶ to 10⁸ per minute.Typically, 10⁸ yeast cells may be transformed per minute, and 10¹⁰-10¹¹bacterial cells may be transformed per minute. In some aspects of theseembodiments, the transformation of cells results in at least 90% viablecells, or 95% viable cells, and up to 99% viable cells.

In some aspects of these embodiments, the FTEP device is manufactured byinjection molding from crystal styrene, cyclo-olefin polymer, orcyclo-olefin co-polymer, and in some aspects of this embodiment theelectrodes are fabricated from stainless steel. In some aspects of theseembodiments, the FTEP devices are fabricated as multiple FTEP devices inparallel on a single substrate where the FTEP devices are then separatedfor use.

In some embodiments of the automated multi-module cell processing systemof which the FTEP is a part, the nucleic acids in the one or morereceptacles comprise a vector backbone and an editing cassette (e.g., anoligonucleotide designed to direct nuclease-directed editing uponexpression in the cell), and the automated multi-module cell editinginstrument further comprises a nucleic acid assembly module. In someaspects, the nucleic acid assembly module comprises a magnet, and insome aspects, the nucleic acid assembly module is configured to performnucleic acid assembly using a single, isothermal reaction. In otheraspects, the nucleic acid assembly module is configured to perform anamplification and/or ligation method. In some aspects, the nucleic acidassembly module also comprises means for isolating, washing,concentrating, diluting and/or resuspending the assembled nucleic acids.

In some embodiments of the automated multi-module cell editinginstrument of which the FTEP is a part, the editing module and therecovery module are combined.

In some embodiments, the automated multi-module cell editing instrumentcomprising the FTEP may further comprise a growth module configured togrow the cells, and in some implementations, the growth module measuresoptical density of the growing cells, either continuously or atintervals. In some implementations, a processor controlling theinstrument is configured to adjust growth conditions in the growthmodule such that the cells reach a target optical density at a timerequested by a user. Further, in some embodiments, the user may beupdated regarding growth process, e.g. through a user interface of theautomated multi-module cell editing instrument or through a portablecomputing device application in communication with the automatedmulti-module cell editing instrument.

In some embodiments, the automated multi-module cell editing instrumentcomprising the FTEP also comprises a reagent cartridge with one or morereceptacles configured to receive cells and one or more receptaclesconfigured to receive nucleic acids.

In some embodiments, the automated multi-module cell editing instrumentcomprising the FTEP also comprises a reagent cartridge with one or morereceptacles configured to receive both cells and nucleic acids. Further,the reagent cartridge may also contain some or all reagents required forcell editing. In some implementations, the reagents contained within thereagent cartridge are locatable by a script read by the processor, andin some implementations, the reagent cartridge includes reagents and isprovided in a kit. In some embodiments, the FTEP device (e.g.,transformation module) is contained within the reagent cartridge.

Some embodiments of the automated multi-module cell editing instrumentfurther comprise a filtration module configured to exchange the liquidmedium in which the cells are suspended and/or concentrate the cells. Inspecific aspects, the filtration system can also be used to render thecells electrocompetent.

In other embodiments, an automated multi-module cell editing instrumentis provided, where the automated multi-module cell editing instrumentcomprises a housing configured to house some or all of the modules; areceptacle configured to receive cells; at least one receptacleconfigured to receive a vector backbone and an editing cassette; anucleic acid assembly module configured to a) assemble the vectorbackbone and editing cassette, and b) de-salt assembled nucleic acidsafter assembly; a growth module configured to grow the cells and measureoptical density (OD) of the cells; a filtration module configured toconcentrate the cells and render the cells electrocompetent; atransformation module comprising an FTEP device to introduce theassembled nucleic acids into the cells; a combination recovery andediting module configured to allow the cells to recover afterelectroporation in the transformation module and to allow the assemblednucleic acids to edit nucleic acids in the cells; and a processorconfigured to operate the automated multi-module cell editing instrumentbased on user input and/or selection of an appropriate controllerscript.

In some implementations, the FTEP device is provided as part of areagent cartridge, which also comprises a plurality of reagentreservoirs and a script readable by a processor for dispensing reagentslocated in the plurality of reagent reservoirs and controlling theflow-through electroporation device.

In some aspects, the growth module includes a temperature-controlledrotating growth vial, a motor assembly to spin the vial, aspectrophotometer for measuring, e.g., OD in the vial, and a processorto accept input from a user and control the growth rate of the cells.The growth module may automatically measure the OD of the growing cellsin the rotating growth vial continuously or at set intervals, andcontrol the growth of the cells to a target OD and a target time asspecified by the user. That is, the methods and devices described hereinprovide a feedback loop that monitors cell growth in real time, andadjusts parameters (e.g., the temperature of the rotating growth vial)in real time to reach the target OD at a target time specified by auser.

Systems for using the automated multi-module cell editing instrument toimplement genomic editing operations within cells are also provided.These systems optionally include one or more interfaces between theinstrument and other devices or receptacles for cell preparation,nucleic acid preparation, selection of edited cell populations,functional analysis of edited cell populations, storage of edited cellpopulations, and the like.

In addition, methods for using the automated multi-module cell editinginstrument containing an FTEP device are provided. In some methods,electrocompetent cells are provided directly to the instrument andtransferred to a transformation module. In some methods, cells aretransferred to a growth module, where they are grown to a desiredoptical density. The cells are then transferred from the growth vial toa filtration module where they are concentrated and optionally renderedelectrocompetent. The cells are then transferred to a the FTEP device.

In some aspects, assembled nucleic acids for transformation are provideddirectly to the instrument, and transferred to a transformation module.In some aspects, nucleic acids, such as a vector backbone and one ormore oligonucleotide editing cassettes, are transferred to a nucleicacid assembly module either simultaneously or sequentially with the cellintroduction or preparation. In this aspect, nucleic acids areassembled, de-salted (e.g., through a liquid exchange or osmosis), andtransferred to an FTEP device to be electroporated into theelectrocompetent cells. Electroporation (e.g., transformation ortransfection) takes place in the FTEP device, then the transformed cellsare transferred to a recovery/editing module that optionally includesselection of the cells containing the one or more genomic edits. Afterrecovery, editing, and/or selection, the cells may be retrieved and useddirectly for research or stored for further research, or subjected toanother round (or multiple rounds) of genomic editing by repeating theediting steps within the instrument.

Also provided are cell libraries created using an automated multi-modulecell editing instrument, where the instrument comprises: a housing; areceptacle configured to receive cells and one or morerationally-designed nucleic acids comprising sequences to facilitatenuclease-directed genome editing events in the cells; an FTEP device forintroduction of the nucleic acid(s) into the cells; an editing modulefor allowing the nuclease-directed genome editing events to occur in thecells, and a processor configured to operate the automated multi-modulecell editing instrument based on user input, wherein thenuclease-directed genome editing events created by the automatedinstrument result in a cell library comprising individual cells withrationally-designed edits.

In some aspects, the cell library created using the instruments andmethods of the disclosure comprises a saturation mutagenesis celllibrary. In some aspects, the cell library created using the instrumentsand methods of the disclosure comprises a promoter swap cell library. Inother aspects, the cell library created using the instruments andmethods of the disclosure comprises a terminator swap cell library. Inyet other aspects, the cell library created using the instruments andmethods of the disclosure comprises a single nucleotide polymorphism(SNP) swap cell library. In yet other aspects, the cell library createdusing the instruments and methods of the disclosure comprises a promoterswap cell library. In some implementations, the library created usingthe instruments and methods of the disclosure comprises at least 100,000edited cells, and in yet other implementations, the library createdusing the instruments and methods of the disclosure comprises at least1,000,000 edited cells. In some implementations, the nuclease-directedgenome editing is RGN-directed genome editing. In a preferred aspect,the instrument is configured for using an inducible nuclease or guidenucleic acid. The nuclease may be, e.g., chemically induced, virallyinduced, light induced, temperature induced, or heat induced.

In some embodiments that involve recursive editing, the automatedmulti-module cell editing instruments of the disclosure introduce two ormore genome edits into cells, with a single genome edit added to thegenomes of the cell population for each cycle. Alternatively, someaspects the automated multi-module cell editing instruments of thepresent disclosure are useful for providing two or more edits per cellin a cell population per cycle, three or more edits per cell in a cellpopulation, five or more edits per cell in a population, or 10 or moreedits per cell in a single cycle for a cell population. In eitherscenario, one or more sequential cycles of editing may be performed.

In specific embodiments, the automated multi-module cell editinginstrument is able to provide an editing efficiency of at least 10% ofthe cells introduced to the editing module per cycle, preferably anediting efficiency of at least 20% of the cells introduced to theediting module per cycle, more preferably an editing efficiency of atleast 25% of the cells introduced to the editing module per cycle, stillmore preferably an editing efficiency of at least 30% of the cellsintroduced to the editing module automated multi-module cell editinginstrument per cycle, yet more preferably an editing efficiency of atleast 40% of the cells introduced to the editing module per cycle andeven more preferably 50%, 60%, 70%, 80%, 90% or more of the cellsintroduced to the editing module per cycle.

Other features, advantages, and aspects will be described below in moredetail.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of the specification, illustrate one or more embodiments and,together with the description, explain these embodiments. Theaccompanying drawings have not necessarily been drawn to scale. Anydimensions illustrated in the accompanying graphs and figures are forillustration purposes only and may or may not represent actual orpreferred values or dimensions. Where applicable, some or all featuresmay not be illustrated to assist in the description of underlyingfeatures. In the drawings:

FIGS. 1A and 1B depict plan and perspective views of an exampleembodiment of an automated multi-module cell editing instrument for themultiplexed genome editing of multiple cells using a replaceablecartridge(s) as a part of the instrument.

FIGS. 2A and 2B depict side and front views of the automatedmulti-module cell editing instrument of FIGS. 1A and 1B. FIGS. 2C and 2Ddepict a second example chassis of an automated multi-module cellediting instrument.

FIG. 3 depicts an example combination nucleic acid assembly module andpurification module for use in an automated multi-module cell editinginstrument.

FIG. 4A is an illustration of a top view of one embodiment of the FTEPdevices of the disclosure. FIG. 4B is an illustration of the top view ofa cross section of the embodiment of the device shown in FIG. 4A. FIG.4C is an illustration of a side view of a cross section of theembodiment of the device shown in FIGS. 4A and 4B. FIG. 4D is anillustration of a top view of another embodiment of the FTEP devices ofthe disclosure. FIG. 4E is an illustration of the top view of a crosssection of the embodiment of the device shown in FIG. 4D. FIG. 4F is anillustration of a side view of a cross section of the embodiment of thedevice shown in FIGS. 4D and 4E. FIG. 4G is an illustration of a topview of yet another embodiment of the FTEP devices of the disclosure.FIG. 4H is an illustration of the top view of a cross section of theembodiment of the device shown in FIG. 4G. FIG. 4I is an illustration ofa side view of a cross section of the embodiment of the device shown inFIGS. 4G and 4H.

FIG. 5A is an illustration of the top view of a cross section of afurther embodiment of the FTEP devices described herein with separateinlets for the cells and the exogenous materials. FIG. 5B is anillustration of the top view of a cross section of the embodiment of thedevice shown in FIG. 5A. FIG. 5C is an illustration of a side view of across section of the embodiment of the device shown in FIG. 5B. FIG. 5Dis an illustration of a side view of a cross section of a variation onthe embodiment of the device shown in FIGS. 5A and 5B. FIG. 5E is anillustration of a side view of a cross section of another variation onthe embodiment of the device shown in FIGS. 5C and 5D. FIG. 5F is anillustration of the top view of a cross section of yet anotherembodiment of the FTEP devices of the disclosure where the FTEPcomprises two separate inlets for the cells and the exogenous materials.FIG. 5G is an illustration of a top view of a cross section of theembodiment of the device shown in FIG. 5F. FIG. 5H is an illustration ofa side view of a cross section of the embodiment of the device shown inFIGS. 5F and 5G.

FIG. 6 is an illustration of a top view of a cross section of yet anadditional embodiment of the FTEP devices of the disclosure, hereincluding flow focusing of fluid from the input channels.

FIG. 7A is an illustration of a top view of a cross section of a firstmultiplexed embodiment of the FTEP devices of the disclosure. FIG. 7B isan illustration of a top view of a cross section of a second multiplexedembodiment of the devices of the disclosure. FIG. 7C is an illustrationof a top view of a cross section of a third multiplexed embodiment ofthe devices of the disclosure. FIG. 7D is an illustration of a top viewof a cross section of a fourth multiplexed embodiment of the devices ofthe disclosure. FIG. 7E is an illustration of a top view of a crosssection of a fifth multiplexed embodiment of the devices of thedisclosure.

FIG. 8A is an illustration of a top view of yet another embodiment ofthe FTEP devices of the disclosure where the electrodes are placed oneither end of the narrowed region of the flow channel rather than oneither side and defining the narrowed region of the flow channel. FIG.8B is an illustration of the top view of a cross section of theembodiment of the device shown in FIG. 8A. FIG. 8C is an illustration ofa side view of a cross section of the embodiment of the device shown inFIGS. 8A and 8B. FIG. 8D is an illustration of a side view of a crosssection of the bottom half of the embodiment of the devices shown inFIGS. 8A, 8B and 8C. FIG. 8E is an illustration of a side view of across section of a variation of the embodiment of the devices shown inFIGS. 8A-8D where here the electrodes are positioned on the bottom ofthe FTEP device, on the opposite surface from the inlet and outlet. FIG.8F is an illustration of a top view of yet another embodiment of theFTEP devices of the disclosure. FIG. 8G an illustration of the top viewof a cross section of the embodiment of the device shown in FIG. 8F.FIG. 8H is an illustration of a side view of a cross section of onevariation of the embodiment of the device shown in FIGS. 8F and 8G.

FIG. 8I is an illustration of a top view of an embodiment of the FTEPdevices of the disclosure. FIG. 8J is an illustration of the top view ofa cross section of the embodiment of the device shown in FIG. 8I wherein this embodiment the FTEP device comprises a filter. FIG. 8K is anillustration of the top view of a cross section of a variation of theembodiment of the device shown in FIGS. 8I and 8J. FIG. 8L is anillustration of a side view of a cross section of the embodiment of thedevices shown in FIGS. 8I-8K. FIG. 8M is an illustration of a side viewof a cross section of the bottom half of the embodiment of the devicesshown in FIGS. 8I-8L. FIG. 8N is an illustration of a top view of yetanother embodiment of the FTEP devices of the disclosure. FIG. 8O is anillustration of the top view of a cross section of the embodiment of thedevice shown in FIG. 8N. FIG. 8P is an illustration of a side view of across section of the embodiment of the device of the disclosure shown inFIGS. 8N-8O. FIG. 8Q is an illustration of a side view of a crosssection of a variation on the embodiment of the device shown in FIGS.8N-8O. FIG. 8R is an illustration of a side view of a cross section ofanother variation on the embodiment of the device shown in FIGS. 8N-8Q.

FIG. 8S is an illustration of the top view of a cross section of yetanother embodiment of the FTEP devices of the disclosure. FIG. 8T is anillustration of the top view of a cross section of the embodiment of thedevice shown in FIG. 8S. FIG. 8U is an illustration of a side view of across section of the embodiment of the device shown in FIGS. 8S and 8T.

FIG. 9A is an illustration of a side view of a cross section of anotherembodiment of the FTEP devices of the disclosure. FIG. 9B is anillustration of the top view of a cross section of the embodiment of thedevice shown in FIG. 9A. FIG. 9C is an illustration of a top view of across section of an embodiment of an FTEP device with a flow focusingfeature.

FIGS. 10A through 10C are top perspective, bottom perspective, andbottom views, respectively, of a flow-through electroporation devicethat may be part of a stand-alone FTEP module or as one module in anautomated multi-module cell processing system. FIG. 10D shows scanningelectromicrographs of the FTEP units depicted in FIG. 10C. FIG. 10Eshows scanning electromicrographs of filters 1070 and 1502 depicted asblack bars in FIGS. 10B and 10C. FIG. 10F depicts (i) the electrodesbefore insertion into the FTEP device; (ii) an electrode; and (iii) theelectrode inserted into an electrode channel with the electrode andelectrode channel adjacent to the flow channel. FIG. 10G shows twoscanning electromicrographs of two different configurations of theaperture where the electrode channel meets the flow channel.

FIGS. 11A-11B depict an exploded view and a top view, respectively, ofan example wash cartridge for use in an automated multi-module cellediting instrument. FIGS. 11C-11E depict an example reagent cartridgefor use in an automated multi-module cell editing instrument.

FIGS. 12A-12C provide a functional block diagram and two perspectiveviews of an example filtration module for use in an automatedmulti-module cell editing instrument. FIG. 12D is a perspective view ofan example filter cartridge for use in an automated multi-module cellediting instrument.

FIGS. 13A-13C depict example cell growth module components for use in anautomated multi-module cell editing instrument.

FIG. 14 is a flow chart of an example method for automated multi-modulecell editing.

FIG. 15A is a flow diagram of a first example workflow for automatedediting of bacterial cells by an automated multi-module cell editinginstrument. FIG. 15B is a flow diagram of a second example workflow forautomated editing of a bacterial cells by an automated multi-module cellediting instrument. FIG. 15C is a flow diagram of an example workflowfor automated cell editing of yeast cells by an automated multi-modulecell editing instrument.

FIG. 16 illustrates an example graphical user interface for providinginstructions to and receiving feedback from an automated multi-modulecell editing instrument.

FIG. 17A is a functional block system diagram of another exampleembodiment of an automated multi-module cell editing instrument for themultiplexed genome editing of multiple cells. FIG. 17B is a functionalblock system diagram of yet another example embodiment of an automatedmulti-module cell editing instrument for the recursive, multiplexedgenome editing of multiple cells.

FIG. 18 is an example control system for use in an automatedmulti-module cell editing instrument.

FIG. 19A is a bar graph showing the results of electroporation of E.coli using a device of the disclosure and a comparator electroporationdevice. FIG. 19B is a bar graph showing uptake, cutting, and editingefficiencies of E. coli cells transformed via an FTEP as describedherein benchmarked against a comparator electroporation device.

FIG. 20 is a bar graph showing the results of electroporation of S.cerevisiae using an FTEP device of the disclosure and a comparatorelectroporation method.

FIG. 21 shows a graph of FTEP flow and pressure versus elapsed time(top), as well as a simple depiction of the pressure system and FTEP(bottom).

It should be understood that the drawings are not necessarily to scale,and that like reference numbers refer to like features.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The description set forth below in connection with the appended drawingsis intended to be a description of various, illustrative embodiments ofthe disclosed subject matter. Specific features and functionalities aredescribed in connection with each illustrative embodiment; however, itwill be apparent to those skilled in the art that the disclosedembodiments may be practiced without each of those specific features andfunctionalities. Moreover, all of the functionalities described inconnection with one embodiment are intended to be applicable to theadditional embodiments described herein except where expressly stated orwhere the feature or function is incompatible with the additionalembodiments. For example, where a given feature or function is expresslydescribed in connection with one embodiment but not expressly mentionedin connection with an alternative embodiment, it should be understoodthat the feature or function may be deployed, utilized, or implementedin connection with the alternative embodiment unless the feature orfunction is incompatible with the alternative embodiment.

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and sequencing technology,which are within the skill of those who practice in the art. Suchconventional techniques include synthesis, assembly, hybridization andligation of polynucleotides, and detection of hybridization using alabel. Specific illustrations of suitable techniques can be had byreference to the examples herein. However, other equivalent conventionalprocedures can, of course, also be used. Such conventional techniquesand descriptions can be found in standard laboratory manuals such asGreen, et al., Eds. (1999), Genome Analysis: A Laboratory Manual Series(Vols. I-IV); Weiner, Gabriel, Stephens, Eds. (2007), Genetic Variation:A Laboratory Manual; Dieffenbach, Dveksler, Eds. (2003), PCR Primer: ALaboratory Manual; Bowtell and Sambrook (2003), DNA Microarrays: AMolecular Cloning Manual; Mount (2004), Bioinformatics: Sequence andGenome Analysis; Sambrook and Russell (2006), Condensed Protocols fromMolecular Cloning: A Laboratory Manual; and Sambrook and Russell (2002),Molecular Cloning: A Laboratory Manual (all from Cold Spring HarborLaboratory Press); Stryer, L. (1995) Biochemistry (4th Ed.) W.H.Freeman, New York N.Y.; Gait, “Oligonucleotide Synthesis: A PracticalApproach” 1984, IRL Press, London; Nelson and Cox (2000), Lehninger,Principles of Biochemistry 3^(rd) Ed., W. H. Freeman Pub., New York,N.Y.; Berg et al. (2002) Biochemistry, 5^(th) Ed., W.H. Freeman Pub.,New York, N.Y.; Cell and Tissue Culture: Laboratory Procedures inBiotechnology (Doyle & Griffiths, eds., John Wiley & Sons 1998);Mammalian Chromosome Engineering—Methods and Protocols (G. Hadlaczky,ed., Humana Press 2011); Essential Stem Cell Methods, (Lanza andKlimanskaya, eds., Academic Press 2011), all of which are hereinincorporated in their entirety by reference for all purposes.CRISPR-specific techniques can be found in, e.g., Genome Editing andEngineering From TALENs and CRISPRs to Molecular Surgery, Appasani andChurch, 2018; and CRISPR: Methods and Protocols, Lindgren andCharpentier, 2015; both of which are herein incorporated in theirentirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “an oligo” refers toone or more oligos that serve the same function, to “the methods”includes reference to equivalent steps and methods known to thoseskilled in the art, and so forth. That is, unless expressly specifiedotherwise, as used herein the words “a,” “an,” “the” carry the meaningof “one or more.” Additionally, it is to be understood that terms suchas “left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,”“length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,”“outer” that may be used herein merely describe points of reference anddo not necessarily limit embodiments of the present disclosure to anyparticular orientation or configuration. Furthermore, terms such as“first,” “second,” “third,” etc., merely identify one of a number ofportions, components, steps, operations, functions, and/or points ofreference as disclosed herein, and likewise do not necessarily limitembodiments of the present disclosure to any particular configuration ororientation.

Furthermore, the terms “approximately,” “proximate,” “minor,” andsimilar terms generally refer to ranges that include the identifiedvalue within a margin of 20%, 10% or preferably 5% in certainembodiments, and any values therebetween.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs.

All publications (including patents, published applications, andnon-patent literature) mentioned herein are incorporated by referencefor all purposes, including but not limited to the purpose of describingand disclosing devices, systems, and methods that may be used ormodified in connection with the presently described methods, modules,instruments, and systems.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the disclosure. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the disclosure, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the disclosure.

Reference throughout the specification to “one embodiment” or “anembodiment” means that a particular feature, structure, orcharacteristic described in connection with an embodiment is included inat least one embodiment of the subject matter disclosed. Thus, theappearance of the phrases “in one embodiment” or “in an embodiment” invarious places throughout the specification is not necessarily referringto the same embodiment.

Further, the particular features, structures or characteristics may becombined in any suitable manner in one or more embodiments. Further, itis intended that embodiments of the disclosed subject matter covermodifications and variations thereof.

INTRODUCTION AND OVERVIEW

In selected embodiments, the automated multi-module cell editinginstruments and systems comprising FTEP devices described herein can beused in multiplexed genome editing in living cells, as well as inmethods for constructing libraries of edited cell populations. Theautomated multi-module cell editing instruments disclosed herein can beused for a variety of genome editing techniques, and in particular withnuclease-directed genome editing techniques. The automated multi-modulecell editing instruments of the disclosure provide novel methods andmodules for introducing nucleic acid sequences into live cells to targetgenomic sites. The methods include constructing libraries comprisingvarious classes of genomic edits to coding regions, non-coding regions,or both. The automated multi-module cell editing instruments areparticularly suited to introducing genome edits to multiple cells in asingle cycle, thereby generating libraries of cells having one or moregenome edits in an automated, multiplexed fashion. The automatedmulti-module cell editing instruments are also suited to introduce twoor more edits, e.g., edits to different target genomic sites inindividual cells of a cell population.

Whether one or many, the genome edits are preferably rationally-designededits; that is, nucleic acids that are designed and created to introducespecific edits to target regions within a cell's genome. The sequencesused to facilitate genome-editing events include sequences that assistin guiding nuclease cleavage, introducing a genome edit to a region ofinterest, and/or both. The sequences may also include an edit to aregion of the cell's genome to allow the specific rationally-designededit in the cell's genome to be tracked. Such methods of introducingedits into cells are taught, e.g., in U.S. Pat. No. 9,982,278, entitled“CRISPR enabled multiplexed genome engineering,” and U.S. Pat. Nos.10,017,760, 10,017,760, entitled “Methods for generating barcodedcombinatorial libraries.”

Nuclease-Directed Genome Editing

In selected embodiments, the automated multi-module cell editinginstruments comprising the FTEP devices described herein utilize anuclease-directed genome editing system. Multiple differentnuclease-based systems exist for editing an organism's genome, and eachcan be used in either single editing systems, sequential editing systems(e.g., using different nuclease-directed systems sequentially to providetwo or more genome edits in a cell) and/or recursive editing systems,(e.g. utilizing a single nuclease-directed system to introduce two ormore genome edits in a cell). Exemplary nuclease-directed genome editingsystems are described herein, although a person of skill in the artwould recognize upon reading the present disclosure that otherenzyme-directed editing systems are also supported by the automatedmulti-module cell editing instruments and FTEP devices of theillustrative embodiments. That is, it should be noted that the automatedinstruments and systems as set forth herein can use the introducednucleases to cleave the genome and introduce an edit into a targetgenomic region.

In particular aspects of the illustrative embodiments, the nucleaseediting system is an inducible system that allows control of the timingof the editing. The inducible system may include inducible expression ofthe nuclease, inducible expression of the editing cassette(s), or both.The ability to modulate nuclease activity can reduce off-target cleavageand facilitate precise genome engineering. Further, inducible systemsare useful when selecting for edited cells as described in U.S. Ser.Nos. 62/718,449 filed 14 Aug. 2018; and 62/724,851, filed 30 Aug. 2018,both of which are incorporated by reference in their entirety-.

In certain aspects, cleavage by a nuclease can be also be used in theautomated multi-module cell editing instruments described and claimed toselect cells with a genomic edit at a target region. For example, cellsthat have been subjected to a genomic edit using an RNA-directednuclease that removes a particular nuclease recognition site or nucleaserecognition site can be selected using the automated multi-module cellediting instruments and systems of the illustrative embodiments byexposing the cells to a nuclease following such edit. The DNA in thecells without the genome edit will be cleaved and subsequently will havelimited growth and/or perish, whereas the cells that received the genomeedit removing the nuclease recognition site will not be affected by thesubsequent exposure to the nuclease.

The promoters driving transcription of one or more components of thenucleic acid-guided nuclease editing system (e.g., one or both of thenuclease and guide nucleic acid) may be inducible, and an induciblesystem is likely employed if selection is to be performed. A number ofgene regulation control systems have been developed for the controlledexpression of genes in plant, microbe, and animal cells, includingmammalian cells, including the pL promoter (induced by heat inactivationof the CI857 repressor), the pBAD promoter (induced by the addition ofarabinose to the cell growth medium), and the rhamnose induciblepromoter (induced by the addition of rhamnose to the cell growthmedium). Other systems include the tetracycline-controlledtranscriptional activation system (Tet-On/Tet-Off, Clontech, Inc. (PaloAlto, Calif.); Bujard and Gossen, PNAS, 89(12):5547-5551 (1992)), theLac Switch Inducible system (Wyborski et al., Environ Mol Mutagen,28(4):447-58 (1996); DuCoeur et al., Strategies 5(3):70-72 (1992); U.S.Pat. No. 4,833,080), the ecdysone-inducible gene expression system (Noet al., PNAS, 93(8):3346-3351 (1996)), the cumate gene-switch system(Mullick et al., BMC Biotechnology, 6:43 (2006)), and thetamoxifen-inducible gene expression (Zhang et al., Nucleic AcidsResearch, 24:543-548 (1996)) as well as others.

The cells that can be transformed or transfected using the FTEP devicesand edited using the automated multi-module cell editing instrumentsinclude any prokaryotic, archaeal or eukaryotic cell. For example,prokaryotic cells for use with the present illustrative embodiments canbe gram positive bacterial cells, e.g., Bacillus subtilis, or gramnegative bacterial cells, e.g., E. coli cells. Eukaryotic cells for usewith the automated multi-module cell editing instruments of theillustrative embodiments include any plant cells and any animal cells,e.g. fungal cells, insect cells, amphibian cells, nematode cells, ormammalian cells.

In selected embodiments, the automated multi-module cell editinginstruments described herein perform zinc-finger nuclease genomeediting. Zinc-finger nucleases (ZFNs) are artificial restriction enzymesgenerated by fusing a zinc finger DNA-binding domain to a DNA-cleavagedomain. Zinc finger domains can be engineered to target-specific regionsin an organism's genome. (Urnov et al., Nature Reviews Genetics,11:636-646 (2010); International Patent Application Publication WO2003/087341 A2 to Carroll et al., entitled “Targeted ChromosomalMutagenesis Using Zinc Finger Nucleases,” filed Jan. 22, 2003). Usingthe endogenous DNA repair machinery of an organism, ZFNs can be used toprecisely alter a target region of the genome. ZFNs can be used todisable dominant mutations in heterozygous individuals by producingdouble-strand breaks (“DSBs”) in the DNA in the mutant allele, whichwill, in the absence of a homologous template, be repaired bynon-homologous end-joining (NHEJ). NHEJ repairs DSBs by joining the twoends together and usually produces no mutations, provided that the cutis clean and uncomplicated. (Durai et al., Zinc finger nucleases:custom-designed molecular scissors for genome engineering of plant andmammalian cells, Nucleic Acids Res., 33(18):5978-90 (2005)). This repairmechanism can be used to induce errors in the genome via indels orchromosomal rearrangement, often rendering the gene products coded atthat location non-functional.

Multiple pairs of ZFNs can also be used to completely remove entirelarge segments of genomic sequence (Lee et al., Genome Res., 20 (1):81-9 (2009); and US Patent Application Publication 2011/0082093 A1 toGregory et al. entitled “Methods and Compositions for TreatingTrinucleotide Repeat Disorders,” filed Jul. 28, 2010). Expanded CAG/CTGrepeat tracts are the genetic basis for more than a dozen inheritedneurological disorders including Huntington's disease, myotonicdystrophy, and several spinocerebellar ataxias. It has been demonstratedin human cells that ZFNs can direct DSBs to CAG repeats and shrink therepeat from long pathological lengths to short, less toxic lengths(Mittelman, et al., Zinc-finger directed double-strand breaks within CAGrepeat tracts promote repeat instability in human cells, PNAS USA, 106(24): 9607-12 (2009); and US Patent Application Publication 2013/0253040A1 to Miller et al. entitled “Methods and Compositions for TreatingHuntington's Disease,” filed Feb. 28, 2013).

In another embodiment, the automated multi-module cell editing modules,instruments, and systems described herein perform meganuclease genomeediting. Meganucleases were identified in the 1990s, and subsequent workhas shown that they are particularly promising tools for genome editing,as they are able to efficiently induce homologous recombination,generate mutations in coding or non-coding regions of the genome, andalter reading frames of the coding regions of genomes. (See, e.g.,Epinat, et al., Nucleic Acids Research, 31(11): 2952-62 (2003); and U.S.Pat. No. 8,921,332 to Choulika et al. entitled “Chromosomal ModificationInvolving the Induction of Double-stranded DNA Cleavage and HomologousRecombination at the Cleavage Site,” issued Dec. 30, 2014.) The highspecificity of meganucleases gives them a high degree of precision andmuch lower cell toxicity than other naturally occurring restrictionenzymes.

In yet another embodiment, the automated multi-module cell editingmodules, instruments and systems described herein perform transcriptionactivator-like effector nuclease editing. Transcription activator-likeeffector nucleases (TALENs) are restriction enzymes that can beengineered to cut specific sequences of DNA. They are made by fusing aTAL effector DNA-binding domain to a DNA cleavage domain (a nucleasewhich cuts DNA strands). Transcription activator-like effector nucleases(TALENs) can be engineered to bind to practically any desired DNAsequence, so when combined with a nuclease, DNA can be cut at specificlocations. (See, e.g., Miller, et al., Nature Biotechnology, 29 (2):143-8 (2011); Boch, Nature Biotech., 29(2): 135-6 (2011); InternationalPatent Application Publication WO 2010/079430 A1 to Bonas et al.entitled “Modular DNA-binding Domains and Methods of Use,” filed Jan.12, 2010; International Patent Application Publication WO 2011/072246 A2to Voytas et al. entitled “TAL Effector-Mediated DNA Modification,”filed Dec. 10, 2010).

Alternatively, DNA can be introduced into a genome in the presence ofexogenous double-stranded DNA fragments using homology dependent repair(HDR). The dependency of HDR on a homologous sequence to repair DSBs canbe exploited by inserting a desired sequence within a sequence that ishomologous to the flanking sequences of a DSB which, when used as atemplate by HDR system, leads to the creation of the desired changewithin the genomic region of interest.

Like ZFNs, TALENs can edit genomes by inducing DSBs. The TALEN-createdsite-specific DSBs at target regions are repaired through NHEJ or HDR,resulting in targeted genome edits. TALENs can be used to introduceindels, rearrangements, or to introduce DNA into a genome through NHEJin the presence of exogenous double-stranded DNA fragments.

In other embodiments, the genome editing of the automated multi-modulecell editing instruments of the illustrative embodiments utilizeclustered regularly interspaced short palindromic repeats (CRISPR)techniques, in which RNA-guided nucleases (RGNs) are used to editspecific target regions in an organism's genome. By delivering the RGNcomplexed with a synthetic guide RNA (gRNA) into a cell, the cell'sgenome can be cut at a desired location, allowing edits to the targetregion of the genome. The guide RNA helps the RGN proteins recognize andcut the DNA of the target genome region. By manipulating the nucleotidesequence of the guide RNA, the RGN system may be programmed to targetany DNA sequence for cleavage.

The RGN system used with the automated multi-module cell editinginstruments of the illustrative embodiments can perform genome editingusing any RNA-guided nuclease system with the ability to both cut andpaste at a desired target genomic region. In certain aspects, theRNA-guided nuclease system may use two separate RNA molecules as a gRNA,e.g., a CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA).In other aspects, the gRNA may be a single gRNA that includes both thecrRNA and tracrRNA sequences.

In certain aspects, the genome editing both introduces a desired DNAchange to a target region and removes the proto-spacer motif (PAM)region from the target region, thus precluding any additional editing ofthe genome at that target region, e.g., upon exposure to a RNA-guidednuclease complexed with a synthetic gRNA complementary to the targetregion. (See, e.g., U.S. Pat. Nos. 9,982,278 and 10,017,760 both ofwhich are incorporated herein in their entirety.) In this aspect, afirst editing event can be, e.g., an RGN-directed editing event or ahomologous recombination event, and cells having the desired edit can beselected using an RGN complexed with a synthetic gRNA complementary tothe target region. Cells that did not undergo the first editing eventwill be cut, and thus will not continue to be viable under appropriateselection criteria. The cells containing the desired mutation will notbe cut, as they will no longer contain the necessary PAM site, and willcontinue to grow and propagate in the automated multi-module cellediting instrument.

When an RGN system is used for selection, it is primarily the cuttingactivity that is needed; thus the RNA-guided nuclease protein system caneither be the same as used for editing, or may be a RGN system that isefficient in cutting using a particular PAM site, but not necessarilyefficient in editing at the site. One important aspect of the nucleaseused for selection is the recognition of the PAM site that is replacedusing the editing approach of the previous genome editing operation.

In yet another embodiment, the genome editing of the automatedmulti-module cell editing instruments of the illustrative embodimentscan utilize homologous recombination methods including the cre-loxtechnique and the FRET technique. Site-specific homologous recombinationdiffers from general homologous recombination in that short specific DNAsequences, which are required for the recombinase recognition, are theonly sites at which recombination occurs. Site-specific recombinationrequires specialized recombinases to recognize the sites and catalyzethe recombination at these sites. A number of bacteriophage- andyeast-derived site-specific recombination systems, each comprising arecombinase and specific cognate sites, have been shown to work ineukaryotic cells for the purpose of DNA integration and are thereforeapplicable for use in the present invention, and these include thebacteriophage P1 Cre/lox, yeast FLP-FRT system, and the Dre system ofthe tyrosine family of site-specific recombinases. Such systems andmethods of use are described, for example, in U.S. Pat. Nos. 7,422,889;7,112,715; 6,956,146; 6,774,279; 5,677,177; 5,885,836; 5,654,182; and4,959,317, which are incorporated herein by reference to teach methodsof using such recombinases. Other systems of the tyrosine family such asbacteriophage lambda Int integrase, HK2022 integrase, and in additionsystems belonging to the separate serine family of recombinases such asbacteriophage phiC31, R4Tp901 integrases are known to work in mammaliancells using their respective recombination sites, and are alsoapplicable for use in the present invention. Exemplary methodologies forhomologous recombination are described in U.S. Pat. Nos. 6,689,610;6,204,061; 5,631,153; 5,627,059; 5,487,992; and 5,464,764, each of whichis incorporated by reference in its entirety.

Instrument Architecture

FIGS. 1A and 1B depict one example of an automated multi-module cellediting instrument 100 utilizing cartridge-based source materials (e.g.,reagents, enzymes, nucleic acids, wash solutions, etc.). The instrument100, for example, may be designed as a desktop instrument for use withina laboratory environment. The instrument 100 may incorporate a mixtureof reusable and disposable elements for performing various stagedoperations in conducting automated genome cleavage and/or editing incells. Cartridge-based source materials, for example, may be positionedin designated areas on a deck 102 of the instrument 100 for access by arobotic handling instrument 108. As illustrated in FIG. 1B, the deck 102may include a protection sink such that contaminants spilling, dripping,or overflowing from any of the modules of the instrument 100 arecontained within a lip of the protection sink.

Turning to FIG. 1A, the instrument 100, in some implementations,includes a reagent cartridge 104 for introducing DNA samples and othersource materials to the instrument 100, an FTEP device 110 c (here as apart of reagent cartridge 104), a wash cartridge 106 for introducingeluent and other source materials to the instrument 100, and a robotichandling system 108 for moving materials between modules (for example,modules 110 a, 110 b, and 110 c) cartridge receptacles (for example,receptacles of cartridges 104 and 106), and storage units (e.g., units112, 114, 116, and 118) of the instrument 100 to perform automatedgenome cleavage and/or editing. Upon completion of processing of a cellsupply, in some embodiments, cell output may be transferred by the robothandling instrument 108 to a storage unit or receptacle placed in, e.g.,reagent cartridge 104 or wash cartridge 106 for temporary storage andlater retrieval.

The robotic handling system 108, for example, may include an airdisplacement pump 120 to transfer liquids from the various reagentreservoirs of the cartridges 104, 106 to the various modules 110 a-110 cand to the storage units (112, 114, 116 or 118). In other embodiments,the robotic handling system 108 may include a pick and place head (notillustrated) to transfer containers of source materials (e.g., tubes orvials) from the reagent cartridge 104 and/or the wash cartridge 106 tothe various modules 110 a-110 c. In some embodiments, one or morecameras or other optical sensors (not shown) confirm proper movement andposition of the robotic handling apparatus along gantry 122.

In some embodiments, the robotic handling system 108 uses disposabletransfer tips provided in a transfer tip supply 116 (e.g., pipette tiprack) to transfer source materials, reagents (e.g., nucleic acids,enzymes, buffers), and cells within the instrument 100. Used transfertips, for example, may be discarded in a solid waste unit 112. In someimplementations, the solid waste unit 112 contains a kicker to removetubes, tips, vials, and/or filters from the pick and place head ofrobotic handling system 108. For example, as illustrated the robotichandling system 108 includes a filter pickup head 124.

In some implementations, the instrument 100 is controlled by aprocessing system 126 such as the processing system 1810 of FIG. 18. Theprocessing system 126 may be configured to operate the instrument 100based on user input. For example, user input may be received by theinstrument 100 through a touch screen control display 128. Theprocessing system 126 may control the timing, duration, temperature andother operations of the various modules 110 a-110 c of the instrument100. Turning to FIG. 1B, the processing system 126 may be connected to apower source 150 for the operation of the instrument 100.

Returning to FIG. 1A, the reagent cartridge 104, as illustrated,includes sixteen reservoirs (a matrix of 5×3 reservoirs, plus anadditional reservoir) and a flow-through FTEP device 110 c (e.g.,transformation modules as described in detail below in relation to FIGS.4A-4I, 5A-5G, 6, 7A-7E, 8A-8U, 9A-9C and 10A-10G). The wash cartridge106 may be configured to accommodate large tubes or reservoirs to store,for example, wash solutions, or solutions that are used often throughoutan iterative process. Further, in some embodiments the wash cartridge106 may include a number of smaller tubes, vials, or reservoirs toretain smaller volumes of, e.g., source media as well as a receptacle orrepository for edited cells. For example, the wash cartridge 106 may beconfigured to remain in place when two or more reagent cartridges 104are sequentially used and replaced. Although the reagent cartridge 104and wash cartridge 106 are shown in FIG. 1A as separate cartridges, inother embodiments, the contents of the wash cartridge 106 may beincorporated into the reagent cartridge 104. In further embodiments,three or more cartridges may be loaded into the automated multi-modulecell editing instrument 100. In certain embodiments, the reagentcartridge 104, wash cartridge 106, and other components of the modules110 a-110 c in the automated multi-module cell editing instrument 100are packaged together in a kit.

The wash and reagent cartridges 104, 106, in some implementations, aredisposable kits provided for use in the automated multi-module cellediting instrument 100. For example, the user may open and position eachof the reagent cartridge 104 and the wash cartridge 106 within a chassisof the automated multi-module cell editing instrument prior toactivating cell processing. Example chassis are discussed in furtherdetail below in relation to FIGS. 2A through 2D.

Components of the cartridges 104, 106, in some implementations, aremarked with machine-readable indicia, such as bar codes, for recognitionby the robotic handling system 108. For example, the robotic handlingsystem 108 may scan containers within each of the cartridges 104, 106 toconfirm contents. In other implementations, machine-readable indicia maybe marked upon each cartridge 104, 106, and the processing system of theautomated multi-module cell editing instrument 100 may identify a storedmaterials map based upon the machine-readable indicia. (See, e.g.,element 1112 of FIG. 11B and element 1130 of FIG. 11D.)

In some embodiments, the wash cartridge 106 (FIG. 1A) is in someembodiments a wash cartridge such as that illustrated in FIGS. 11A-11B.The cartridge 1100 includes a pair of containers 1102 a, b, a set offour small tubes 1104 a, b, c, d, and a larger tube 1106 held in acartridge body 1108. One or more of the containers 1102 a, b, and tubes1104 a, b, c, d and 1106, in some embodiments, is sealed with apierceable foil for access by an automated liquid handling system, suchas a sipper or pipettor. In other embodiments, one or more of thecontainers 1102 a, b, and tubes 1104 a, b, c, d, and 1106 includes asealable access gasket. The top of one or more of the containers 1102 a,b, and tubes 1104 a, b, c, d, and 1106, in some embodiments, is markedwith machine-readable indicia (not illustrated) for automatedidentification of the contents.

In some embodiments, containers 1102 a, b contain wash solutions. Thewash solution may be a same or different wash solutions. In someexamples, wash solutions may contain, e.g., buffer, buffer and 10%glycerol, 80% ethanol.

In some implementations, a cover 1120 secures the containers 1102 a, band tubes 1104 a, b, c, d and 1106 within the cartridge body 1108.Turning to FIG. 11B, the cover 1120 may include apertures for access toeach of the containers 1102 a, b and tubes 1104 a, b, c, d and 1106.Further, the cover 1120 may include machine-readable indicia 1112 foridentifying the type of cartridge (e.g., accessing a map of thecartridge contents). Alternatively, apertures may be marked separatelywith the individual contents.

In some embodiments, the reagent cartridge 104 of FIG. 1A is a reagentcartridge such as that illustrated in FIG. 11C. FIG. 11C shows a reagentcartridge 1122 including a set of eighteen tubes or vials 1140; however,the embodiment shown in FIG. 11C does not include an FTEP device.Looking at FIG. 11E, reagent cartridge includes sixteen tubes or vials1126 a-p and an FTEP device 1124, held in a cartridge body 1122. One ormore of the tubes or vials 1140 (FIG. 11C) or 1126 a-p (FIG. 11E), insome embodiments, is sealed with pierceable foil for access by anautomated liquid handling system, such as a sipper or pipettor. In otherembodiments such as that shown in FIG. 11E, one or more of the tubes orvials 1126 a-1126 p includes a sealable access gasket. The top of eachof the small tubes or vials 1126 a-1126 p, in some embodiments, ismarked with machine-readable indicia (not illustrated) for automatedidentification of the contents. The machine-readable indicia may includea bar code, QR code, or other machine-readable coding. Other automatedmeans for identifying a particular container can include color coding,symbol recognition (e.g., text, image, icon, etc.), and/or shaperecognition (e.g., a relative shape of the container). Rather than beingmarked upon the vessel itself, in some embodiments, an upper surface ofthe cartridge body and/or the cartridge cover may containmachine-readable indicia for identifying contents. The small tubes orvials may each be of a same size. Alternatively, multiple volumes oftubes or vials may be provided in the reagent cartridge 1120. In anillustrative example, each tube or vial may be designed to hold between2 and 20 mL, between 4 and 10 mL, or about 5 mL. In some embodimentswhere only small volumes of some reagents are required, tube inserts maybe used to accommodate small (e.g., microfuge) tubes in a largerreceptacle.

In an illustrative example, the tubes or vials 1126 a-1126 p may eachhold one the following materials: a vector backbone, oligonucleotides,reagents for nucleic acid assembly, a user-supplied cell sample, aninducer agent, magnetic beads in buffer, ethanol, an antibiotic for cellselection, reagents for eluting cells and nucleic acids, an oil overlay,other reagents, and cell growth and/or recovery media.

In some implementations, a cover 1120 as seen in FIG. 11D secures thetubes or vials 1140 within the cartridge body 1122 of FIG. 11C. Turningto FIG. 11D, the cover 1120 may include apertures for access to each ofthe small tubes or vials 1126. Three large apertures 1132 are outlinedin a bold band to indicate positions to add user-supplied materials. Theuser-supplied materials, for example, may include a vector backbone,oligonucleotides, and a cell sample. Further, the cover 1120 may includemachine-readable indicia 1130 for identifying the type of cartridge(e.g., accessing a map of the cartridge contents). Alternatively, eachaperture may be marked separately with the individual contents. In someimplementations, to ensure positioning of user-supplied materials, thevials or tubes provided for filling in the lab environment may haveunique shapes or sizes such that the cell sample vial or tube only fitsin the cell sample aperture, the oligonucleotides vial or tube only fitsin the oligonucleotides aperture, and so on.

Turning back to FIG. 1A, also illustrated is the robotic handling system108 including the gantry 122. In some examples, the robotic handlingsystem 108 may include an automated liquid handling system such as thosemanufactured by Tecan Group Ltd. of Mannedorf, Switzerland, HamiltonCompany of Reno, Nev. (see, e.g., WO2018015544A1 to Ott, entitled“Pipetting device, fluid processing system and method for operating afluid processing system”), or Beckman Coulter, Inc. of Fort Collins,Colo. (see, e.g., US20160018427A1 to Striebl et al., entitled “Methodsand systems for tube inspection and liquid level detection”). Therobotic handling system 108 may include an air displacement pipettor120. The reagent cartridges 104, 106 allow for particularly easyintegration with the liquid handling instrumentation of the robotichandling system 108 such as air displacement pipettor 120. In someembodiments, only the air displacement pipettor 120 is moved by thegantry 122 and the various modules 110 a-110 c and cartridges 104, 106remain stationary. Pipette tips may be provided in a pipette transfertip supply 116 for use with the air displacement pipettor 120.

In some embodiments, an automated mechanical motion system (actuator)(not shown) additionally supplies XY axis motion control or XYZ axismotion control to one or more modules 110 a-110 c and/or cartridges 104,106 of the automated multi-module cell editing instrument 100. Usedpipette tips, for example, may be placed by the robotic handling systemin a waste repository 112. For example, an active module may be raisedto come into contact-accessible positioning with the robotic handlingsystem or, conversely, lowered after use to avoid impact with therobotic handling system as the robotic handling system is movingmaterials to other modules within the automated multi-module cellediting instrument 100.

The automated multi-module cell editing instrument 100, in someimplementations, includes the FTEP device 110 c (e.g., transformation ortransfection module) included in the reagent cartridge 104. Aflow-through electroporation connection bridge 132, for example, isengaged with the flow-through electroporation device after the cells andnucleic acids are transferred into the device via an input channel. Thebridge 132 provides both a liquid-tight seal and an electricalconnection to the electrodes, as well as control for conductingelectroporation within the FTEP device 110 c. For example, theelectroporation connection bridge 132 may be connected to FTEP controls134 within an electronics rack 136 of the automated multi-module cellediting instrument 100.

In some implementations, the automated multi-module cell editinginstrument 100 includes dual cell growth modules 110 a, 110 b. The cellgrowth modules 110 a, 110 b, as illustrated each include a rotating cellgrowth vial 130 a, 130 b. At least one of the cell growth modules 110 a,110 b may additionally include an integrated filtration module (notillustrated). In alternative embodiments, a filtration module or a cellwash and concentration module may instead be separate from cell growthmodules 110 a, 110 b (e.g., as described in relation to cell growthmodule 1710 a and filtration module 1710 b of FIGS. 17A and 17B). Thecell growth modules 110 a, 110 b, for example, may each include thefeatures and functionalities discussed in relation to the cell growthmodule 1300 of FIGS. 13A-C.

A filtration portion of one or both of the cell growth modules 110 a,110 b, in some embodiments, uses replaceable filters stored in a filtercassette 118. For example, the robotic handling system may include thefilter pick-up head 124 to pick up and engage filters for use with oneor both of the cell growth modules 110 a, 110 b. The filter pick-up headtransfers a filter to the growth module, pipettes up the cells from thegrowth module, then washes and renders the cells electrocompetent. Themedium from the cells, and the wash fluids are disposed in waste module114.

In some implementations, automated multi-module cell editing instrument100 includes a nucleic acid assembly and purification function (e.g.,nucleic acid assembly module) for combining materials provided in thereagent cartridge 104 into an assembled nucleic acid for cell editing.Further, a desalting or purification operation purifies the assemblednucleic acids and de-salts the buffer such that the nucleic acids aremore efficiently electroporated into the cells. The nucleic acidassembly and purification feature may include a reaction chamber or tubereceptacle (not shown) and a magnet (not shown).

Although the example instrument 100 is illustrated as including aparticular arrangement of modules 110, this implementation is forillustrative purposes only. For example, in other embodiments, more orfewer modules 110 may be included within the instrument 100, anddifferent modules may be included such as, e.g., a module for cellfusion to produce hybridomas and/or a module for expression and/orprotein production. Further, certain modules may be replicated withincertain embodiments, such as the duplicate cell growth modules 110 a,110 b of FIG. 1A.

In some embodiments, the cells are modified prior to introduction ontothe automated multi-module cell editing instrument. For example,bacterial cells of interest may harbor a λ red system to facilitategenome repair, and/or the cells may harbor an antibiotic resistancegene, so they may be selected easily. FIGS. 2A through 2D illustrateexample chassis 200 and 230 for use in desktop versions of an automatedmulti-module cell editing instrument. For example, the chassis 200 and230 may have a width of about 24-48 inches, a height of about 24-48inches and a depth of about 24-48 inches. Each of the chassis 200 and230 may be designed to hold multiple modules and disposable suppliesused in automated cell processing. Further, each chassis 200 and 230 maymount a robotic handling system for moving materials between modules.

FIGS. 2A and 2B depict a first example chassis 200 of an automatedmulti-module cell editing instrument. As illustrated, the chassis 200includes a cover 202 having a handle 204 and hinges 206 a-206 c forlifting the cover 202 and accessing an interior of the chassis 200. Acooling grate 214 may allow for air flow via an internal fan (notshown). Further, the chassis 200 is lifted by adjustable feet 220. Thefeet 220 a-220 c, for example, may provide additional air flow beneaththe chassis 200. A control button 216, in some embodiments, allows forsingle-button automated start and/or stop of cell processing within thechassis 200.

Inside the chassis 200, in some implementations, a robotic handlingsystem 208 is disposed along a gantry 210 above materials cartridges 212a, 212 b. Control circuitry, liquid handling tubes, air pump controls,valves, thermal units (e.g., heating and cooling units) and othercontrol mechanisms, in some embodiments, are disposed below a deck ofthe chassis 200, in a control box region 218.

Although not illustrated, in some embodiments a display screen may bepositioned upon a front face of the chassis 200, for example covering aportion of the cover 202. The display screen may provide information tothe user regarding a processing status of the automated multi-modulecell editing instrument. In another example, the display screen mayaccept inputs from the user for conducting the cell processing.

FIGS. 2C and 2D depict a second example chassis 230 of an automatedmulti-module cell editing instrument. The chassis 230, as illustrated,includes a transparent door 232 with a hinge 234. For example, the doormay swing to the left of the page to provide access to a work area ofthe chassis. The user, for example, may open the transparent door 232 toload supplies, such as reagent cartridges and wash cartridges, into thechassis 230.

In some embodiments, a front face of the chassis 230 further includes adisplay (e.g., touch screen display device) 236 illustrated to the rightof the door 232. The display 236 may provide information to the userregarding a processing status of the automated multi-module cell editinginstrument. In another example, the display 236 may accept inputs fromthe user, e.g., for pausing or conducting the cell processing.

An air grate 238 on a right face of the chassis 230 may provide for airflow within a work area (e.g., above the deck) of the chassis 230 (e.g.,above a deck). A second air grate 240 on a left of the chassis 230 mayprovide for air flow within a control box region 242 (e.g., below thedeck) of the chassis 230. Although not illustrated, in some embodiments,feet such as the feet 220 a-220 c of the chassis 200 may raise thechassis 230 above a work surface, providing for further air flow.

Inside the chassis 230, in some implementations, a robotic handlingsystem 248 is disposed along a gantry 250 above cartridges 252 a, 252 b,material supplies 254 a, 254 b (e.g., pipette tips and filters), andmodules (e.g., dual growth vials, FTEP device, nucleic acid assemblymodule (not shown)). Control circuitry, liquid handling tubes, air pumpcontrols, valves, and other control mechanisms, in some embodiments, aredisposed below a deck of the chassis 230, in the control box region 242.

In some embodiments, a liquid waste unit 246 is mounted to the leftexterior wall of the chassis 230. The liquid waste unit 246, forexample, may be mounted externally to the chassis 230 to avoid potentialcontamination and to ensure prompt emptying and replacement of theliquid waste unit 246.

Nucleic Acid Assembly Module

Certain embodiments of the automated multi-module cell editinginstruments of the present disclosure include a nucleic acid assemblymodule instrument. The nucleic acid assembly module is configured toaccept and assemble the nucleic acids necessary to facilitate thedesired genome editing events. The nucleic acid assembly module may alsobe configured to accept the appropriate vector backbone for vectorassembly and subsequent electroporation into the cells of interest.

In general, the term “vector” refers to a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked.Vectors include, but are not limited to, nucleic acid molecules that aresingle-stranded, double-stranded, or partially double-stranded; nucleicacid molecules that include one or more free ends, no free ends (e.g.circular); nucleic acid molecules that include DNA, RNA, or both; andother varieties of polynucleotides known in the art. One type of vectoris a “plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,where virally-derived DNA or RNA sequences are present in the vector forpackaging into a virus (e.g. retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses). Viral vectors also include polynucleotidescarried by a virus for transfection into a host cell. Certain vectorsare capable of autonomous replication in a host cell into which they areintroduced (e.g. bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively-linked.Such vectors are referred to herein as “expression vectors.” Commonexpression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. Additional vectors include fosmids, phagemids, andsynthetic chromosomes.

Recombinant expression vectors can include a nucleic acid in a formsuitable for transformation, and for some nucleic acid sequences,translation and expression of the nucleic acid in a host cell, whichmeans that the recombinant expression vectors include one or moreregulatory elements—which may be selected on the basis of the host cellsto be used for expression—that are operatively-linked to the nucleicacid sequence to be expressed. Within a recombinant expression vector,“operably linked” is intended to mean that the nucleotide sequence ofinterest is linked to the regulatory element(s) in a manner that allowsfor transcription, and for some nucleic acid sequences, translation andexpression of the nucleotide sequence (e.g. in an in vitrotranscription/translation system or in a host cell when the vector isintroduced into the host cell). Appropriate recombination and cloningmethods are disclosed in U.S. patent application Ser. No. 10/815,730,entitled “Recombinational Cloning Using Nucleic Acids HavingRecombination Sites” published Sep. 2, 2004 as US 2004-0171156 A1, thecontents of which are herein incorporated by reference in their entiretyfor all purposes.

In some embodiments, a regulatory element is operably linked to one ormore elements of a targetable nuclease system so as to drivetranscription, and for some nucleic acid sequences, translation andexpression of the one or more components of the targetable nucleasesystem.

In addition, the polynucleotide sequence encoding the nucleicacid-guided nuclease can be codon optimized for expression in particularcells, such as prokaryotic or eukaryotic cells. Eukaryotic cells can beyeast, fungi, algae, plant, animal, or human cells. Eukaryotic cells maybe those of or derived from a particular organism, such as a mammal,including but not limited to human, mouse, rat, rabbit, dog, ornon-human mammal including non-human primate. In addition oralternatively, a vector may include a regulatory element operably likedto a polynucleotide sequence, which, when transcribed, forms a guideRNA.

The nucleic acid assembly module can be configured to perform a widevariety of different nucleic acid assembly techniques in an automatedfashion. Nucleic acid assembly techniques that can be performed in thenucleic acid assembly module of the disclosed automated multi-modulecell editing instruments include, but are not limited to, those assemblymethods that use restriction endonucleases, including PCR, BioBrickassembly (U.S. Pat. No. 9,361,427 to Hillson entitled “Scar-lessMulti-part DNA Assembly Design,” issued Jun. 7, 2016), Type IIS cloning(e.g., GoldenGate assembly; European Patent Application Publication EP 2395 087 A1 to Weber et al. entitled “System and Method of ModularCloning,” filed Jul. 6, 2010), and Ligase Cycling Reaction (de Kok S,ACS Synth Biol., 3(2):97-106 (2014); Engler, et al., PLoS One,3(11):e3647 (2008); U.S. Pat. No. 6,143,527 to Pachuk et al. entitled“Chain Reaction Cloning Using a Bridging Oligonucleotide and DNALigase,” issued Nov. 7, 2000). In other embodiments, the nucleic acidassembly techniques performed by the disclosed automated multi-modulecell editing instruments are based on overlaps between adjacent parts ofthe nucleic acids, such as Gibson Assembly®, CPEC, SLIC, Ligase Cyclingetc. Additional assembly methods include gap repair in yeast (Bessa,Yeast, 29(10):419-23 (2012)), gateway cloning (Ohtsuka, Curr PharmBiotechnol, 10(2):244-51 (2009); U.S. Pat. No. 5,888,732 to Hartley etal., entitled “Recombinational Cloning Using Engineered RecombinationSites,” issued Mar. 30, 1999; U.S. Pat. No. 6,277,608 to Hartley et al.entitled “Recominational Cloning Using Nucleic Acids HavingRecombination Sites,” issued Aug. 21, 2001), and topoisomerase-mediatedcloning (Udo, PLoS One, 10(9):e0139349 (2015); U.S. Pat. No. 6,916,632B2 to Chestnut et al. entitled “Methods and Reagents for MolecularCloning,” issued Jul. 12, 2005). These and other nucleic acid assemblytechniques are described, e.g., in Sands and Brent, Curr Protoc MolBiol., 113:3.26.1-3.26.20 (2016); Casini et al., Nat Rev Mol Cell Biol.,(9):568-76 (2015); Patron, Curr Opinion Plant Biol., 19:14-9 (2014)).

The nucleic acid assembly module is temperature controlled dependingupon the type of nucleic acid assembly used in the automatedmulti-module cell editing instrument. For example, when PCR is utilizedin the nucleic acid assembly module, the module will have athermocycling capability allowing the temperatures to cycle betweendenaturation, annealing and extension. When single temperature assemblymethods are utilized in the nucleic acid assembly module, the modulewill have the ability to reach and hold at the temperature thatoptimizes the specific assembly process being performed. Thesetemperatures and the duration for maintaining these temperatures can bedetermined by a preprogrammed set of parameters executed by a script, ormanually controlled by the user using the processing system of theautomated multi-module cell editing instrument.

In one embodiment, the nucleic acid assembly module is a module toperform assembly using a single, isothermal reaction, such as thatillustrated in FIG. 3. Certain isothermal assembly methods can combinesimultaneously up to 15 nucleic acid fragments based on sequenceidentity. The assembly method provides, in some embodiments, nucleicacids to be assembled which include an approximate 20-40 base overlapwith adjacent nucleic acid fragments. The fragments are mixed with acocktail of three enzymes—an exonuclease, a polymerase, and aligase-along with buffer components. Because the process is isothermaland can be performed in a 1-step or 2-step method using a singlereaction vessel, isothermal assembly reactions are ideal for use in anautomated multi-module cell editing instrument. The 1-step method allowsfor the assembly of up to five different fragments using a single stepisothermal process. The fragments and the master mix of enzymes arecombined and incubated at 50° C. for up to one hour. For the creation ofmore complex constructs with up to fifteen fragments or forincorporating fragments from 100 bp up to 10 kb, typically the 2-step isused, where the 2-step reaction requires two separate additions ofmaster mix; one for the exonuclease and annealing step and a second forthe polymerase and ligation steps.

FIG. 3 illustrates an example nucleic acid assembly module 300 withintegrated purification. The nucleic acid assembly module 300 includes achamber 302 having an access gasket 304 for transferring liquids to andfrom the nucleic acid assembly module 300 (e.g., via a pipette orsipper). In some embodiments, the access gasket 304 is connected to areplaceable vial which is positioned within the chamber 302. Forexample, a user or robotic manipulation system may place the vial withinthe nucleic acid assembly module 300 for processing.

The chamber 302 shares a housing 306 with a resistive heater 308. Once asample has been introduced to the chamber 302 of the nucleic acidassembly module 300, the resistive heater 308 may be used to heat thecontents of the chamber 302 to a desired temperature. Thermal rampingmay be set based upon the contents of the chamber 302 (e.g., thematerials supplied through the access gasket 304 via pipettor or sipperunit of the robotic manipulation system). The processing system of theautomated multi-module cell editing instrument may determine the targettemperature and thermal ramping plan. The thermal ramping and targettemperature may be controlled through monitoring a thermal sensor suchas a thermistor 310 included within the housing 306. In a particularembodiment, the resistive heater 308 is designed to maintain atemperature within the housing 306 of between 20° and 80° C., between25° and 75° C., between 37° and 65° C., between 40° and 60° C., between45 and 55° C. or preferably about 50° C.

Purification Module

In some embodiments, when a nucleic acid assembly module is included inthe automated multi-module cell editing instrument, the instrument alsocan include a purification module to remove unwanted components of thenucleic acid assembly mixture (e.g., salts, minerals) and, in certainembodiments, concentrate the assembled nucleic acids. Examples ofmethods for exchanging the liquid following nucleic acid assemblyinclude magnetic beads (e.g., SPRI or Dynal (Dynabeads) by InvitrogenCorp. of Carlsbad, Calif.), silica beads, silica spin columns, glassbeads, precipitation (e.g., using ethanol or isopropanol), alkalinelysis, osmotic purification, extraction with butanol, membrane-basedseparation techniques, filtration etc.

In one aspect, the purification module provides filtration, e.g.,ultrafiltration. For example, a range of microconcentrators fitted withanisotropic, hydrophilic-generated cellulose membranes of varyingporosities is available. In another example, the purification andconcentration involves contacting a liquid sample including theassembled nucleic acids and an ionic salt with an ion exchangerincluding an insoluble phosphate salt, removing the liquid, and elutingthe nucleic acid from the ion exchanger.

In a specific aspect of the purification module, SPRI beads can be usedwhere 0.6-2.0× volumes of SPRI beads can be added to the nucleic acidassembly. The nucleic acid assembly product becomes bound to the SPRIbeads, and the SPRI beads are pelletal by automatically positioning amagnet close to the tube, vessel, or chamber harboring the pellet. Forexample, 0.6-2.0× volumes of SPRI beads can be added to the nucleic acidassembly. The SPRI beads, for example, may be washed with ethanol, andthe bound nucleic acid assembly product is eluted. e.g., in water, Trisbuffer, or 10% glycerol.

in a specific aspect, a magnet is coupled to a linear actuator thatpositions the magnet. In some implementations, the nucleic acid assemblymodule is a combination assembly and purification module designed forintegrated assembly and purification. For example, as discussed above inrelation to the nucleic acid assembly module depicted in FIG. 3, oncesufficient time has elapsed for the nucleic acid assembly reaction totake place, the contents of the chamber 302 (e.g., the nucleic acidassembly reagents and nucleic acids), in some embodiments, are combinedwith magnetic beads (not shown) to activate the purification process.The SPRI beads in buffer are delivered to the contents of the nucleicacid assembly module, for example, by a robotic handling system.Thereafter, a solenoid 312, in some embodiments, is actuated by a magnetto excite the magnetic beads contained within the chamber 302. Thesolenoid, in a particular example, may impart between a 2 pound magneticpull force and a 5 pound pull force, or approximately a 4 pound magneticpull force to the magnetic beads within the chamber 302. The contents ofthe chamber 302 may be incubated for sufficient time for the assembledvector and oligonucleotides to bind to the magnetic beads.

After binding, in some implementations, the bound nucleic acid assemblymix (e.g., nucleic acid assembly reagents+assembled vector andoligonucleotides) is removed from the nucleic acid assembly module andthe nucleic acids attached to the beads are washed one to several timeswith 80% ethanol. Once washed, the nucleic acids attached to the beadsare eluted into buffer and are transferred to the transformation module.That is, in some embodiments, the nucleic acid assembly module andpurification module are combined.

In some implementations, a vial is locked in position in the chamber 302for processing. For example, a user may press the vial beyond a detentin the chamber 302 designed to retain the vial upon engagement with apipettor or sipper. In another example, the user may twist the vial intoposition, thus engaging a protrusion to a corresponding channel andbarring upward movement. A position sensor (not illustrated) may ensureretraction of the vial. The position sensor, in a particular embodiment,is a magnetic sensor detecting engagement between a portion of thechamber 302 and the vial. In other embodiments, the position sensor isan optical sensor detecting presence of the vial at a retractedposition. In embodiments using a channel and protrusion, a mechanicswitch pressed down by the protrusion may detect engagement of the vial

Growth Module

As the nucleic acids are being assembled, the cells may be grown inpreparation for editing. Cell growth can be monitored by optical density(e.g., at OD 600 nm) that is measured in a growth module, and a feedbackloop is used to adjust the cell growth so as to reach a target OD at atarget time. Other measures of cell density and physiological state thatcan be measured include but are not limited to, pH, dissolved oxygen,released enzymes, acoustic properties, and electrical properties.

In some aspects, the growth module includes a culture tube in a shakeror vortexer that is interrogated by a spectrophotometer or fluorimeter.The shaker or vortexer can heat or cool the cells and cell growth ismonitored by real-time absorbance or fluorescence measurements. In oneaspect, the cells are grown at 25° C.-40° C. to an OD600 absorbance of1-10 ODs. The cells may also be grown at temperature ranges from 25°C.-35° C., 25° C.-30° C., 30° C.-40° C., 30° C.-35° C., 35° C.-40° C.,40° C.-50° C., 40° C.-45° C. or 44° C.-50° C. In another aspect, thecells are induced by heating at 42° C.-50° C. or by adding an inducingagent. The cells may also be induced by heating at ranges from 42°C.-46° C., 42° C.-44° C., 44° C.-46° C., 44° C.-48° C., 46° C.-48° C.,46° C.-50° C., or 48° C.-50° C. In some aspects, the cells are cooled to0° C.-10° C. after induction. The cells may also be cooled totemperature ranges of 0° C.-5° C., 0° C.-2° C., 2° C.-4° C., 4° C.-6°C., 6° C.-8° C., 8° C.-10° C., or 5° C.-10° C. after induction.

FIG. 13A shows one embodiment of a rotating growth vial 1300 for usewith a cell growth device, such as cell growth device 1350 illustratedin FIGS. 13B-C. The rotating growth vial 1300, in some implementations,is a transparent container having an open end 1304 for receiving liquidmedia and cells, a central vial region 1306 that defines the primarycontainer for growing cells, a tapered-to-narrowed region 1318 definingat least one light path 1308, 1310, a closed end 1316, and a driveengagement mechanism 1312. The rotating growth vial 1300 may have acentral longitudinal axis 1320 around which the vial 1300 rotates, andthe light paths 1308, 1310 may be generally perpendicular to thelongitudinal axis of the vial. In some examples, first light path 1310may be positioned in the lower narrowed portion of thetapered-to-narrowed region 1318. The drive engagement mechanism 1312, insome implementations, engages with a drive mechanism (e.g., actuator,motor (not shown)) to rotate the vial 1300. The actuator may include adrive shaft 1374 for a drive motor (not shown).

In some embodiments, the rotating growth vial 1300 includes a secondlight path 1308, for example, in the upper tapered region of thetapered-to-narrowed region 1318. In some examples, the walls definingthe upper tapered region of the tapered-to-narrowed region 1318 for thesecond light path 1308 may be disposed at a wider angle relative to thelongitudinal axis 1320 than the walls defining the lower narrowedportion of the tapered-to-narrowed region 1310 for the first light path1310. Both light paths 1308, 1310, for example, may be positioned in aregion of the rotating growth vial 1300 that is constantly filled withthe cell culture (cells+growth media), and is not affected by therotational speed of the growth vial 1300. As illustrated, the secondlight path 1308 is shorter than the first light path 1310 allowing forsensitive measurement of optical density (OD) values when the OD valuesof the cell culture in the vial are at a high level (e.g., later in thecell growth process), whereas the first light path 1310 allows forsensitive measurement of OD values when the OD values of the cellculture in the vial are at a lower level (e.g., earlier in the cellgrowth process).

The rotating growth vial 1300 may be reusable, or preferably, therotating growth vial is consumable. In some embodiments, the rotatinggrowth vial 1300 is consumable and can be presented to the userpre-filled with growth medium, where the vial 1300 is sealed at the openend 1304 with a foil seal. A medium-filled rotating growth vial packagedin such a manner may be part of a kit for use with a stand-alone cellgrowth device or with a cell growth module that is part of an automatedmulti-module cell editing instrument. To introduce cells into the vial,a user need only pipette up a desired volume of cells and use thepipette tip to punch through the foil seal of the vial 1300.Alternatively, of course, an automated instrument may transfer cellsfrom, e.g., a reagent cartridge, to the growth vial. The growth mediummay be provided in the growth vial or may also be transferred from areagent cartridge to the growth vial before the addition of cells. Openend 1304 may include an extended lip 1302 to overlap and engage with thecell growth device 1350 (FIG. 13B). In automated instruments, therotating growth vial 1300 may be tagged with a barcode or otheridentifying means that can be read by a scanner or camera that is partof the processing system 1810 as illustrated in FIG. 18.

In some implementations, the volume of the rotating growth vial 1300 andthe volume of the cell culture (including growth medium) may varygreatly, but the volume of the rotating growth vial 1300 should be largeenough for the cell culture in the growth vial 1300 to get properaeration while the vial 1300 is rotating. In practice, the volume of therotating growth vial 1300 may range from 1-250 ml, 2-100 ml, from 5-80ml, 10-50 ml, or from 12-35 ml. Likewise, the volume of the cell culture(cells+growth media) should be appropriate to allow proper aeration inthe rotating growth vial 1300. Thus, the volume of the cell cultureshould be approximately 10-85% of the volume of the growth vial 800, or15-80% of the volume of the growth vial, or 20-70%, 30-60%, or 40-50% ofthe volume of the growth vial. In one example, for a 35 ml growth vial1300, the volume of the cell culture would be from about 4 ml to about27 ml.

The rotating growth vial 1300, in some embodiments, is fabricated from abio-compatible transparent material, or at least the portion of the vial1300 including the light path(s) is transparent. Additionally, materialfrom which the rotating growth vial 1300 is fabricated should be able tobe cooled to about 0° C. or lower and heated to about 75° C. or higher,such as about 2° C. or to about 70° C., about 4° C. or to about 60° C.,or about 4° C. or to about 55° C. to accommodate both temperature-basedcell assays and long-term storage at low temperatures. Further, thematerial that is used to fabricate the vial is preferably able towithstand temperatures up to 55° C. without deformation while spinning.Suitable materials include glass, polyvinyl chloride, polyethylene,polyamide, polyethylene, polypropylene, polycarbonate, poly(methylmethacrylate) (PMMA), polysulfone, polyurethane, and co-polymers ofthese and other polymers. Preferred materials include polypropylene,polycarbonate, or polystyrene. In some embodiments, the rotating growthvial 800 is inexpensively fabricated by, e.g., injection molding orextrusion.

FIGS. 13 B-C illustrate views of an example cell growth device 1350 thatreceives the rotating growth vial 1300. In some embodiments, the cellgrowth device 1350 rotates to heat or cool the cells or cell growthwithin the vial 1300 to a predetermined temperature range. In someimplementations, the rotating growth vial 1300 can be positioned insidea main housing 1352 with the extended lip 1302 of the vial 1300extending past an upper surface of the main housing 1352. In someaspects, the extended lip 1302 provides a grasping surface for a userinserting or withdrawing the vial 1300 from the main housing 1352 of thedevice 1350. Additionally, when fully inserted into the main housing1352, a lower surface of the extended lip 1302 abuts an upper surface ofthe main housing 1352. In some examples, the main housing 1352 of thecell growth device 1350 is sized such that outer surfaces of therotating growth vial 1300 abut inner surfaces of the main housing 1352thereby securing the vial 1300 within the main housing 1352. In someimplementations, the cell growth device 1350 can include end housings1354 disposed on each side of the main housing 1354 and a lower housing1356 disposed at a lower end of the main housing 1352. In some examples,the lower housing 1356 may include flanges 1358 that can be used toattach the cell growth device 1350 to a temperature control (e.g,heating/cooling) mechanism or other structure such as a chassis of anautomated cell processing system.

As shown in FIG. 13C, the cell growth device 1350, in someimplementations, can include an upper bearing 1360 and lower bearing1362 positioned in main housing 1352 that support the vertical load of arotating growth vial 1300 that has been inserted into the main housing1352. In some examples, the cell growth device 1350 may also include aprimary optical port 1366 and a secondary optical port 1368 that arealigned with the first light path 1310 and second light path 1308 of thevial 1300 when inserted into the main housing 1352. In some examples,the primary and secondary optical ports 1366, 1368 are gaps, openings,or portions of the main housing constructed from transparent materialsthat allow light to pass through the vial 1300 to perform cell growth ODmeasurements. In addition to the optical ports 1366, 1368, the cellgrowth device 1350 may include an emission board 1370 that provides oneor more illumination sources for the light path(s), and detector board1372 to detect the light after the light travels through the cellculture liquid in the rotating growth vial 1300. In one example, theillumination sources disposed on the emission board 1370 may includelight emission diodes (LEDs) or photodiodes that provide illumination atone or more target wavelengths commensurate with the growth mediatypically used in cell culture (whether, e.g., mammalian cells,bacterial cells, animal cells, yeast cells).

In some implementations, the emission board 1370 and/or detector board1372 are communicatively coupled through a wired or wireless connectionto a processing system (e.g., processing system 126, 1720, 1810) thatcontrols the wavelength of light output by the emission board 1370 andreceives and processes the illumination sensed at the detector board1372. The remotely controllable emission board 1370 and detector board1372, in some aspects, provide for conducting automated OD measurementsduring the course of cell growth. For example, the processing system126, 1720 may control the periodicity with which OD measurements areperformed, which may be at predetermined intervals or in response to auser request Further, the processing system 126, 1720 can use the sensordata received from the detector board 1372 to perform real-time ODmeasurements and adjust cell growth conditions (e.g., temperature,speed/direction of rotation).

In some embodiments, the lower housing 1356 may contain a drive motor(not shown) that generates rotational motion that causes the rotatinggrowth vial 1300 to spin within the cell growth device 1350. In someimplementations, the drive motor may include a drive shaft 1374 thatengages a lower end of the rotating growth vial 1300. The drive motorthat generates rotational motion for the rotating growth vial 1300, insome embodiments, is a brushless DC type drive motor with built-in drivecontrols that can be configured to maintain a constant revolution perminute (RPM) between 0 and about 3000 RPM. Alternatively, other motortypes such as a stepper, servo, or brushed DC motors can be used.Optionally, the drive motor may also have direction control to allowreversing of the rotational direction, and a tachometer to sense andreport actual RPM. In other examples, the drive motor can generateoscillating motion by reversing the direction of rotation at apredetermined frequency. In one example, the vial 1300 is rotated ineach direction for one second at a speed of 350 RPM. The drive motor, insome implementations, is communicatively coupled through a wired orwireless communication network to a processing system (e.g., processingsystem 126, 1720) that is configured to control the operation of thedrive motor, which can include executing protocols programmed into theprocessor and/or provided by user input, for example as described inrelation to module controller 1830 of FIG. 18. For example, and thedrive motor can be configured to vary the speed and/or rotationaldirection of the vial 1300 to cause axial precession of the cell culturethereby enhancing mixing in order to prevent cell aggregation andincrease aeration. In some examples, the speed or direction of rotationof the drive motor may be varied based on optical density sensor datareceived from the detector board 1372.

In some embodiments, main housing 1352, end housings 1354 and lowerhousing 1356 of the cell growth device 1350 may be fabricated from arobust material including aluminum, stainless steel, and other thermallyconductive materials, including plastics. These structures or portionsthereof can be created through various techniques, e.g., metalfabrication, injection molding, creation of structural layers that arefused, etc. While in some examples the rotating growth vial 1300 isreusable, in other embodiments, the vial 1300 is preferably isconsumable. The other components of the cell growth device 1350, in someaspects, are preferably reusable and can function as a stand-alonebenchtop device or as a module in an automated multi-module cell editinginstrument.

In some implementations, the processing system that is communicativelycoupled to the cell growth module may be programmed with information tobe used as a “blank” or control for the growing cell culture. A “blank”or control, in some examples, is a vessel containing cell growth mediumonly, which yields 100% transmittance and 0 OD, while the cell samplesdeflect light rays and will have a lower percentage transmittance andhigher OD. As the cells grow in the media and become denser,transmittance decreases and OD increases. The processor of the cellgrowth module, in some implementations, may be programmed to usewavelength values for blanks commensurate with the growth mediatypically used in cell culture (whether, e.g., mammalian cells,bacterial cells, animal cells, yeast cells). Alternatively, a secondspectrophotometer and vessel may be included in the cell growth module,where the second spectrophotometer is used to read a blank at designatedintervals.

To reduce background of cells that have not received a genome edit, thegrowth module may also allow a selection process to enrich for theedited cells. For example, the introduced nucleic acid can include agene, which confers antibiotic resistance or another selectable marker.Alternating the introduction of selectable markers for sequential roundsof editing can also eliminate the background of unedited cells and allowmultiple cycles of the automated multi-module cell editing instrument toselect for cells having sequential genome edits.

Suitable antibiotic resistance genes include, but are not limited to,genes such as ampicillin-resistance gene, tetracycline-resistance gene,kanamycin-resistance gene, neomycin-resistance gene,canavanine-resistance gene, blasticidin-resistance gene,hygromycin-resistance gene, puromycin-resistance gene, andchloramphenicol-resistance gene. In some embodiments, removing dead cellbackground is aided using lytic enhancers such as detergents, osmoticstress, temperature, enzymes, proteases, bacteriophage, reducing agents,or chaotropes. In other embodiments, cell removal and/or media exchangeis used to reduce dead cell background.

Cell Wash and/or Concentration Module

The cell wash and/or concentration module can utilize any method forexchanging the liquids in the automated multi-module cell processinginstrument, and may concentrate the cells or allow them to remain inessentially the same or greater volume of liquid as used in the nucleicacid assembly module. Further, in some aspects, the processes performedin the cell wash module also render the cells electrocompetent, by,e.g., use of glycerol in the wash.

Numerous different methods can be used to wash the cells, includingdensity gradient purification, dialysis, ion exchange columns,filtration, centrifugation, dilution, and the use of beads forpurification.

In some aspects, the cell wash and/or concentration module utilizes acentrifugation device. In other aspects, the cell wash and/orconcentration module utilizes a filtration module. In yet other aspects,beads are coupled to moieties that bind to the cell surface. Thesemoieties include but are not limited to antibodies, lectins, wheat germagglutinin, mutated lysozymes, and ligands.

In other aspects, the cells are engineered to be magnetized, allowingmagnets to pellet the cells after wash steps. The mechanism of cellmagnetization can include but is not limited to ferritin proteinexpression.

The cell wash and/or concentration module, in some implementations, is afiltration module. Turning to FIG. 12A, a block diagram illustratesexample functional units of a filtration module 1200. In someimplementations, a main control 1202 of the filtration module 1200includes a first liquid pump 1204 b to intake wash fluid 1206 and asecond liquid pump 1204 a to remove liquid waste to a liquid waste unit1208 (e.g., such as the liquid waste unit 114 of FIG. 1A or liquid wasteunit 1728 of FIGS. 17A and 17B). A flow sensor 1212 may be disposed on aconnector 1214 to the liquid waste unit 1208 to monitor release ofliquid waste from the filtration module. A valve 1216 (a three-way valveas illustrated) may be disposed on a connector 1218 to the wash fluid1206 in wash cartridge 1210 to selectively connect the wash fluid 1206and the filtration module 1200.

The filtration module 1200, in some implementations, includes a filtermanifold 1220 for filtering and concentrating a cell sample. The filtermanifold 1220 may include one or more temperature sensor(s) 1222 andpressure sensor (s) 1224 to monitor flow and temperature of the washfluid and/or liquid waste. The sensors 1222, 1224, in some embodiments,are monitored and analyzed by a processing system of the automatedmulti-mode cell processing system, such as the processing system 1810 ofFIG. 18. The filter manifold 1220 may include one or more valves 1226and 1126 b for directing flow of the wash fluid and/or liquid waste. Theprocessing system of the automated multi-mode cell processinginstrument, for example, may actuate the valves according to a set ofinstructions for directing filtration by the filtration module 1200.

The filtration module 1200 includes at least one filter 1230. Examplesof filters suitable for use in the filtration module 1200 includemembrane filters, ceramic filters and metal filters. The filter may beused in any shape; the filter may for example be cylindrical oressentially flat. The filter selected for a given operation or a givenworkflow, in some embodiments, depends upon the type of workflow (e.g.,bacterial, yeast, viral, etc.) or the volumes of materials beingprocessed. For example, while flat filters are relatively low cost andcommonly used, filters with a greater surface area, such as cylindricalfilters, may accept higher flow rates. In another example, hollowfilters may demonstrate lower recovery rates when processing smallvolumes of sample (e.g., less than about 10 ml). For example, for usewith bacteria, it may be preferable that the filter used is a membranefilter, particularly a hollow fiber filter. With the term “hollow fiber”is meant a tubular membrane. The internal diameter of the tube, in someexamples, is at least 0.1 mm, more preferably at least 0.5 mm, mostpreferably at least 0.75 mm and preferably the internal diameter of thetube is at most 10 mm, more preferably at most 6 mm, most preferably atmost 1 mm. Filter modules having hollow fibers are commerciallyavailable from various companies, including G.E. Life Sciences(Marlborough, Mass.) and InnovaPrep (Drexel, Mo.) (see, e.g.,US20110061474A1 to Page et al., entitled “Liquid to Liquid BiologicalParticle Concentrator with Disposable Fluid Path”).

In some implementations, the filtration module 1200 includes a filterejection means 1228 (e.g., actuator) to eject a filter 1230 post use.For example, a user or the robotic handling system may push the filter1230 into position for use such that the filter is retained by thefilter manifold 1220 during filtration. After filtration to remove theused filter 1230, the filter ejection actuator 1228 may eject the filter1230, releasing the filter 1230 such that the user or the robotichandling system may remove the used filter 1230 from the filtrationmodule 1200. The used filter 1230, in some examples, may be disposedwithin the solid waste unit 112 of FIGS. 1A-1B, solid waste unit 1718 ofFIGS. 17A and 17B, or returned to a filter cartridge 1240, asillustrated in FIG. 12D.

Turning to FIG. 12D, in some implementations, filters 1230 a, b, c, dprovided in the filter cartridge 1240 disposed within the chassis of theautomated multi-module cell editing instrument are transported to thefiltration module 1200 by a robotic handling system (e.g., the robotichandling system 108 described in relation to FIGS. 1A and 1B, or robotichandling system 1708 of FIGS. 17A and 17B) and positioned within thefiltration module 1200 prior to use.

The filtration module 1200, in some implementations, requires periodiccleaning. For example, the processing system may alert a user whencleaning is required through the user interface of the automatedmulti-module cell editing instrument and/or through a wireless messagingmeans (e.g., text message, email, and/or personal computing deviceapplication). A decontamination filter, for example, may be loaded intothe filtration module 1200 and the filtration module 1200 may be cleanedwith a wash solution and/or alcohol mixture.

In some implementations, the filtration module 1200 is in fluidconnection with a wash cartridge 1210 (such as the wash cartridge 1100of FIG. 11A) containing the wash fluid 1206 via the connector 1218. Forexample, upon positioning by the user of the wash cartridge 1210 withinthe chassis of the automated multi-module cell editing instrument, theconnector 1218 may mate with a bottom inlet of the wash cartridge 1210,creating a liquid passage between the wash fluid 1206 and the filtrationmodule 1200.

Turning to FIGS. 12B and 12C, in some implementations, a dual filterfiltration module 1250 includes dual filters 1252 a and 1252 b disposedover dual wash reservoirs 1254 a and 1254 b. In an example, each filtermay be a hollow core fiber filter having 0.45 um pores and greater than85 cm² area. The wash reservoirs 1254 a and 1254 b, in some examples,may be 50 mL, 100 mL, or over 200 mL in volume. In some embodiments, thewash reservoirs 1254 a and 1254 b are disposed in a wash cartridge 1256,such as the wash or reagent cartridge 1100 of FIG. 11A.

The processing system of the automated multi-module cell editinginstrument, in some implementations, controls actuation of the dualfilters 1252 a and 1252 b in an X (horizontal) and Z (vertical)direction to position the filters 1252 a, 1252 b in the wash reservoirs1254 a and 1254 b. In a particular example, the dual filters 1252 a and1252 b may be move in concert along the X axis but have independent Zaxis range of motion.

As illustrated, the dual filters 1252 a and 1252 b of the filtrationmodule 1250 are connected to a slender arm body 1258. In someembodiments, any pumps and valves of the filtration module 1250 may bedisposed remotely from the body 1258 (e.g., within a floor of thechassis of the automated multi-module cell editing instrument). In thismanner, the filtration module 1250 may replace much bulkier conventionalcommercial filtration modules.

Further, in some embodiments, the filtration module 1250 is in liquidcommunication with a waste purge system designed to release liquid wasteinto a liquid waste storage unit, such as the liquid waste vessel 1208of FIG. 12A or the liquid waste storage unit 114 of FIG. 1A or 1728 ofFIGS. 17A and 17B.

FTEP Module

The FTEP (transformation) module may implement any cell transformationor transfection techniques routinely performed by electroporation.Electroporation is a widely-used method for permeabilization of cellmembranes that works by temporarily generating pores in the cellmembranes with electrical stimulation. The applications ofelectroporation include the delivery of DNA, RNA, siRNA, peptides,proteins, antibodies, drugs or other substances to a variety of cellssuch as mammalian cells (including human cells), plant cells, archea,yeasts, other eukaryotic cells, bacteria, and other cell types.Electrical stimulation may also be used for cell fusion in theproduction of hybridomas or other fused cells. During a typicalelectroporation procedure, cells are suspended in a buffer or mediumthat is favorable for cell survival. For bacterial cell electroporation,low conductance mediums, such as water, glycerol solutions and the like,are often used to reduce the heat production by transient high current.The cells and material to be electroporated into the cells (collectively“the cell sample”) is then placed in a cuvette embedded with two flatelectrodes for an electrical discharge. For example, Bio-Rad (Hercules,Calif.) makes the GENE PULSER XCELL™ line of products to electroporatecells in cuvettes. Traditionally, electroporation requires high fieldstrength.

Generally speaking, microfluidic electroporation—using cell suspensionvolumes of less than approximately 20 ml and as low as 1 μl—allows moreprecise control over a transfection or transformation process andpermits flexible integration with other cell processing tools comparedto bench-scale electroporation devices. Microfluidic electroporationthus provides unique advantages for, e.g., single cell transformation,processing and analysis; multi-unit FTEP device configurations; andintegrated, automated multi-module cell editing and analysis.

The present disclosure provides electroporation devices, modules, andmethods that achieve high efficiency cell electroporation with lowtoxicity where the electroporation devices and systems can be integratedwith other automated cell processing tools. Further, the electroporationdevice of the disclosure allows for multiplexing where two to manyelectroporation units are constructed and used in parallel, and allowsfor particularly easy integration with robotic liquid handlinginstrumentation. Such automated instrumentation includes, but is notlimited to, off-the-shelf automated liquid handling systems from Tecan(Mannedorf, Switzerland), Hamilton (Reno, Nev.), Beckman Coulter (FortCollins, Colo.), etc.

During the electroporation process, it is important to use voltagesufficient for achieving electroporation of material into the cells, butnot too much voltage as too much voltage will decrease cell viability.For example, to electroporate a suspension of a human cell line, 200volts is needed for a 0.2 ml sample in a 4 mm-gap cuvette withexponential discharge from a capacitor of about 1000 g. However, if thesame 0.2 ml cell suspension is placed in a longer container with 2 cmelectrode distance (5 times of cuvette gap distance), the voltagerequired would be 1000 volts, but a capacitor of only 40 μf. ( 1/25 of1000 g) is needed because the electric energy from a capacitor followsthe equation of:E=0.5U²Cwhere E is electric energy, U is voltage and C is capacitance. Thereforea high voltage pulse generator is actually easy to manufacture becauseit needs a much smaller capacitor to store a similar amount of energy.Similarly, it would not be difficult to generate other wave forms ofhigher voltages.

The electroporation devices of the disclosure can allow for a high rateof cell transformation in a relatively short amount of time. The rate ofcell transformation is dependent on the cell type and the number ofcells being transformed. For example, for E. coli, the electroporationdevices can provide a cell transformation rate of 10³ to 10¹² cells persecond, 10⁴ to 10¹⁰ per second, 10⁵ to 10⁹ per second, or 10⁶ to 10⁸ persecond. Typically, 10⁸ yeast cells may be transformed per minute, and 10¹⁰-10¹² bacterial cells may be transformed per minute. Theelectroporation devices also allow transformation of batches of cellsranging from 1 cell to 10¹² cells in a single transformation procedureusing parallel devices.

One embodiment of the FTEP device described herein is illustrated inFIGS. 4A-4C. FIG. 4A shows a planar top view of an FTEP device 400having an inlet 402 for introducing a fluid containing cells andexogenous material to be delivered to the cells into the FTEP device 400and an outlet 404 for removing the transformed cells followingelectroporation. Oval electrodes 408 are positioned so as to define acenter portion of the flow channel (not shown) where the channel narrowsbased on the curvature of the electrodes. FIG. 4B shows a cutaway viewfrom the top of the device 400, with the inlet 402, outlet 404, andelectrodes 408 positioned with respect to a flow channel 406. Note thatthe electrodes 408 define a narrowing of flow channel 406. FIG. 4C showsa side cutaway view of the device 400 with the inlet 402 and inletchannel 412, and outlet 404 and outlet channel 414. The electrodes 408are oval in shape and positioned so that they define a narrowed portionof the flow channel 406.

In the FTEP devices of the disclosure, the toxicity level of thetransformation results in greater than 30% viable cells afterelectroporation, preferably greater than 35%, 40%, 45%, 50%, 55%, 60%,70%, 75%, 80%, 85%, 90%, 95% or even 99% viable cells followingtransformation, depending on the cell type and the nucleic acids beingintroduced into the cells.

The housing of the FTEP device can be made from many materials dependingon whether the FTEP device is to be reused, autoclaved, or isdisposable, including stainless steel, silicon, glass, resin, polyvinylchloride, polyethylene, polyamide, polystyrene, polyethylene,polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers ofthese and other polymers. Similarly, the walls of the channels in thedevice can be made of any suitable material including silicone, resin,glass, glass fiber, polyvinyl chloride, polyethylene, polyamide,polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers ofthese and other polymers. Preferred materials include crystal styrene,cyclo-olefin polymer (COP) and cyclic olephin co-polymers (COC), whichallow the device to be formed entirely by injection molding in one piecewith the exception of the electrodes and, e.g., a bottom sealing film ifpresent.

The FTEP devices described herein (or portions of the FTEP devices) canbe created or fabricated via various techniques, e.g., as entire devicesor by creation of structural layers that are fused or otherwise coupled.For example, for metal FTEP devices, fabrication may include precisionmechanical machining or laser machining; for silicon FTEP devices,fabrication may include dry or wet etching; for glass FTEP devices,fabrication may include dry or wet etching, powderblasting,sandblasting, or photostructuring; and for plastic FTEP devicesfabrication may include thermoforming, injection molding, hot embossing,or laser machining. The components of the FTEP devices may bemanufactured separately and then assembled, or certain components of theFTEP devices (or even the entire FTEP device except for the electrodes)may be manufactured (e.g., using 3D printing) or molded (e.g., usinginjection molding) as a single entity, with other components added aftermolding. For example, housing and channels may be manufactured or moldedas a single entity, with the electrodes (and, e.g., bottom sealing film)later added to form the FTEP unit (see, FIG. 10F (i)). Alternatively,the FTEP device may also be formed in two or more parallel layers, e.g.,a layer with the horizontal channel and filter, a layer with thevertical channels, and a layer with the inlet and outlet ports, whichare manufactured and/or molded individually and assembled followingmanufacture. (See, e.g., FIG. 9A.)

In specific aspects, the FTEP device can be manufactured using a circuitboard as a base, with the electrodes, filter and/or the flow channelformed in the desired configuration on the circuit board, and theremaining housing of the device containing, e.g., the one or more inletand outlet channels and/or the flow channel formed as a separate layerthat is then sealed onto the circuit board. The sealing of the top ofthe housing onto the circuit board provides the desired configuration ofthe different elements of the FTEP devices of the disclosure. Also, twoto many FTEP devices may be manufactured on a single substrate, thenseparated from one another thereafter or used in parallel. In certainembodiments, the FTEP devices are reusable and, in some embodiments, theFTEP devices are disposable. In additional embodiments, the FTEP devicesmay be autoclavable.

The electrodes 408 can be formed from any suitable metal, such ascopper, stainless steel, titanium, aluminum, brass, silver, rhodium,gold or platinum, or graphite. One preferred electrode material is alloy303 (UNS330300) austenitic stainless steel. An applied electric fieldcan destroy electrodes made from of metals like aluminum. If amultiple-use (i.e., non-disposable) flow-through FTEP device isdesired—as opposed to a disposable, one-use flow-through FTEP device—theelectrode plates can be coated with metals resistant to electrochemicalcorrosion. Conductive coatings like noble metals, e.g., gold, can beused to protect the electrode plates.

Additionally, the FTEP devices may comprise push-pull pneumatic means toallow multi-pass electroporation procedures; that is, cells toelectroporated may be “pulled” from the inlet toward the outlet for onepass of electroporation, then be “pushed” from the outlet end of theflow-through FTEP device toward the inlet end to pass between theelectrodes again for another pass of electroporation. This process maybe repeated one to many times.

Depending on the type of cells to be electroporated (e.g., bacterial,yeast, mammalian) and the configuration of the electrodes, the distancebetween the electrodes in the flow channel can vary widely. For example,in the embodiments shown in FIGS. 4A-4I, 5A-5H, 6, and 7A-7E where theelectrodes form a portion of the wall of the flow channel where the flowchannel decreases in width, the distance between the electrodes in theflow channel may be between 10 μm and 5 mm, or between 25 μm and 3 mm,or between 50 μm and 2 mm, or between 75 μm and 1 mm. In otherembodiments such as those depicted in FIGS. 8A-8U, 9A-9C, and 10A-10Dwhere the electrodes are positioned on either end of the channelnarrowing, the distance between the electrodes in the flow channel maybe between 1 mm and 10 mm, or between 2 mm and 8 mm, or between 3 mm and7 mm, or between 4 mm and 6 mm. The overall size of the FTEP device maybe from 3 cm to 15 cm in length, or 4 cm to 12 cm in length, or 4.5 cmto 10 cm in length. The overall width of the FTEP device may be from 0.5cm to 5 cm, or from 0.75 cm to 3 cm, or from 1 cm to 2.5 cm, or from 1cm to 1.5 cm.

The region of the flow channel that is narrowed is typically wide enoughso that at least two cells can fit in the narrowed portion side-by-side.For example, a typical bacterial cell is 1 μm in diameter; thus, thenarrowed portion of the flow channel of the FTEP device used totransform such bacterial cells will be at least 2 μm wide. In anotherexample, if a mammalian cell is approximately 50 μm in diameter, thenarrowed portion of the flow channel of the FTEP device used totransform such mammalian cells will be at least 100 μm wide. That is,the narrowed portion of the FTEP device will not physically contort or“squeeze” the cells being transformed.

In embodiments of the FTEP device where reservoirs are used to introducecells and exogenous material into the FTEP device, the reservoirs rangein volume from 100 μL to 10 mL, or from 500 μL to 75 mL, or from 1 mL to5 mL. The flow rate in the FTEP ranges from 0.1 mL to 5 mL per minute,or from 0.5 mL to 3 mL per minute, or from 1.0 mL to 2.5 mL per minute.The pressure in the FTEP device ranges from 1-30 psi, or from 2-10 psi,or from 3-5 psi.

To avoid different field intensities between the electrodes, theelectrodes should be arranged in parallel. Furthermore, the surface ofthe electrodes should be as smooth as possible without pin holes orpeaks. Electrodes having a roughness Rz of 1 to 10 μm are preferred. Inanother embodiment of the invention, the flow-through electroporationdevice comprises at least one additional electrode which applies aground potential to the FTEP device.

The electrodes are configured to deliver 1-25 Kv/cm, or 5-20 Kv/cm, or10-20 Kv/cm. The further apart the electrodes are, the more voltageneeds to be supplied; in addition, the voltage delivered of coursedepends on the types of cells being porated, the medium in which thecells are suspended, the size of the electroporation channel, and thelength and diameter of the electrodes. There are many different pulseforms that may be employed with the FTEP device, including exponentialdecay waves, square or rectangular waves, arbitrary wave forms, or aselected combination of wave forms. One type of common pulse form is theexponential decay wave, typically made by discharging a loaded capacitorto the cell sample. The exponential decay wave can be made less steep bylinking an inductor to the cell sample so that the initial peak currentcan be attenuated. When multiple waveforms in a specified sequence areused, they can be in the same direction (direct current) or differentdirections (alternating current). Using alternating current can bebeneficial in that two topical surfaces of a cell instead of just onecan be used for molecular transport, and alternating current can preventelectrolysis. The pulse generator can be controlled by a digital oranalog panel. In some embodiments, square wave forms are preferred, andin other embodiments, an initial wave spike before the square wave ispreferred.

The FTEP device may be configured to electroporate cell sample volumesbetween 1 μl to 5 ml, 10 μl to 2 ml, 25 μl to 1 ml, or 50 μl to 750 μl.The medium or buffer used to suspend the cells and material (reagent) tobe electroporated into the cells for the electroporation process may beany suitable medium or buffer, such as MEM, DMEM, IMDM, RPMI, Hanks',PBS and Ringer's solution, where the media may be provided in a reagentcartridge as part of a kit. Further, because the cells must be madeelectrocompetent prior to transformation or transfection, the bufferalso may comprise glycerol or sorbitol, and may also comprise asurfactant. For electroporation of most eukaryotic cells the medium orbuffer usually contains salts to maintain a proper osmotic pressure. Thesalts in the medium or buffer also render the medium conductive. Forelectroporation of very small prokaryotic cells such as bacteria,sometimes water or 10% glycerol is used as a low conductance medium toallow a very high electric field strength. In that case, the chargedmolecules to be delivered still render water-based medium moreconductive than the lipid-based cell membranes and the medium may stillbe roughly considered as conductive particularly in comparison to cellmembranes.

The compound to be electroporated into the cells can be any compoundknown in the art to be useful for electroporation, such as nucleicacids, oligonucleotides, polynucleotides, DNA, RNA, peptides, proteinsand small molecules like hormones, cytokines, chemokines, drugs, or drugprecursors. In the nucleic acid-guided nuclease editing embodiments, thecompounds electroporated into the cells are nucleic acids and proteins.

Another embodiment of the FTEP devices described herein is illustratedin FIGS. 4D-4F. FIG. 4D shows a top planar view of an FTEP device 410having an inlet 402 for introducing a fluid containing cells andexogenous material into the FTEP device 410 and an outlet 404 forremoving the transformed cells following electroporation. Cylindricalelectrodes 408 are positioned so as to define a center portion of theflow channel (not shown) where the flow channel narrows as a result ofthe curvature of the electrodes. FIG. 4E shows a cutaway view from thetop of the FTEP device 410, with the inlet 402, outlet 404, andelectrodes 408 positioned with respect to a flow channel 406. Again,note that the electrodes 408 define a narrowed portion or region of flowchannel 406. FIG. 4F shows a side cutaway view of FTEP device 410 withthe inlet 402 and inlet channel 412, and outlet 404 and outlet channel414. The electrodes 408 are cylindrical and positioned in the flowchannel 406 defining a narrowed portion of the flow channel 406.

Yet another embodiment of the FTEP devices of the disclosure isillustrated in FIGS. 4G-4I. FIG. 4G shows a top planar view of an FTEPdevice 420 having an inlet 402 for introducing a fluid containing cellsand exogenous material into FTEP device 420, and an outlet 404 forremoving the transformed cells following electroporation. Thesemi-cylindrical electrodes 408 are positioned so as to define anarrowed portion of a flow channel (not shown) where the channel narrowsfrom both ends based on the curvature of the electrodes. FIG. 4H shows acutaway view from the top of FTEP device 420, with the inlet 402, outlet404, and electrodes 408 positioned with respect to a flow channel 406.FIG. 4I shows a side cutaway view of FTEP device 420 with inlet 402 andinlet channel 412, and outlet 404 and outlet channel 414. Thesemi-cylindrical electrodes 408 are positioned in the flow channel 406so that they define a narrowed portion of the flow channel 406. Itshould be noted that the devices depicted in FIGS. 4A-4I show theelectrodes positioned substantially mid-way along the flow channel;however, in other aspects of the devices, the electrodes may bepositioned in narrowed regions of the flow channel more toward the inletof the FTEP device or more toward the outlet of the FTEP device.

FIGS. 5A-5E show embodiments of the FTEP devices of the disclosure withseparate inlets for the cells and the exogenous material. FIG. 5A showsa top planar view of an FTEP device 500 having a first inlet 502 forintroducing a fluid containing cells into FTEP device 500; a secondinlet 518 for introducing a fluid containing exogenous materials to beelectroporated into the cells into FTEP device 500; electrodes 508; andan outlet 504 for removing the transformed cells followingelectroporation. Although these embodiments are illustrated withcylindrical electrodes, as shown in FIG. 5A, other shaped electrodeswith a curved edge—e.g., oval, semi-cylindrical, and the like as shownin relation to FIGS. 4A-4I—may be used to define the flow channel. FIG.5B shows a cutaway view from the top of FTEP device 500, with the firstinlet 502, second inlet 518, outlet 504, and electrodes 508 positionedwith respect to the flow channel 506.

FIG. 5C shows a cutaway view of the embodiment of FTEP device 500 withthe first inlet 502 and second inlet 518 positioned as shown in FIGS. 5Aand 5B. In FIG. 5C, the first inlet channel 512 and second inlet channel522 meet independently with flow channel 506, and the liquid (cells andmaterial to be porated or delivered to the cells) flows through the flowchannel 506 to the outlet channel 514 and outlet 504 where thetransformed cells are removed from the FTEP device. The electrodes 508are positioned in the flow channel 506 so that they define a narrowedportion of the flow channel 506. FIG. 5D shows a side cutaway view of avariation 510 on the embodiment of the FTEP device 500 depicted in FIGS.5A and 5B. Here, the first inlet channel 512 and second inlet channel524 intersect with the flow channel 506 at a three-way junction, and theliquid (cells and material to be porated or delivered to the cells)flows through the flow channel 506 to the outlet channel 514 and outlet504 where the transformed cells are removed from the FTEP device. Theelectrodes 508 are positioned in the flow channel 506 defining anarrowed portion of the flow channel 506. FIG. 5E shows a first sidecutaway view 520 of a yet another variation of the FTEP device 500 shownin FIGS. 5A and 5B. Here, the first inlet channel 512 and second inletchannel 526 intersect at a junction where the cells and exogenousmaterials mix prior to introduction of the combined fluids to the flowchannel 506. The fluids flow through the flow channel 506 to the outletchannel 514 and outlet 504 where the transformed cells are removed fromthe FTEP device. Electrodes 508 are positioned in the flow channel 506so that they define a narrowed portion of the flow channel 506.

FIGS. 5F-5H show another embodiment of the FTEP devices of thedisclosure with separate inlets for the cells and the exogenousmaterial. FIG. 5F shows a top planar view of an electroporation device530 having a first inlet 502 for introducing a fluid containing cells, asecond outlet 518 for introducing exogenous materials to beelectroporated into the cells, and an outlet 504 for removing thetransformed cells following electroporation. The electrodes 508 arepositioned between the first inlet 502 where the cells are introducedinto the FTEP device and the second inlet 518 where the exogenousmaterials are introduced into the FTEP device. FIG. 5G shows a cutawayview from the top of the FTEP device 530, with the first inlet 502,second inlet 518, and outlet 504, and with electrodes 508 positionedbetween the first inlet channel 502 and the second inlet channel 518,where the electrodes 508 form a narrowed portion of flow channel 506.FIG. 5H shows a side cutaway view of FTEP device 530 with the firstinlet 502 where the cells are introduced into the FTEP device and firstinlet channel 512, the second inlet 518 where the exogenous materialsare introduced into the FTEP device and second inlet channel 532, and anoutlet channel 514 and outlet 504 where the transformed cells areremoved from the FTEP device. The electrodes 508 are positioned in theflow channel 506 defining a narrow portion of the flow channel 506 andare positioned between the first inlet channel 512 and the second inletchannel 532 such that the material to be introduced into the cells isadded to the fluid comprising the cells after the cells have beenelectroporated.

FIG. 6 illustrates an FTEP device in which the flow of the fluidintroduced into the flow channel from the input channel(s) is focused,e.g., using an immiscible fluid such as an oil or a stream of air tonarrow the stream of the fluid containing the cells and the exogenousmaterials as it passes by the electrodes. FIG. 6 shows a cutaway viewfrom the top of the FTEP device 600, with the inlet 602, outlet 604, andthe electrodes 608 positioned between the first inlet channel 602 andoutlet 604. The flow focusing 630 is effected by an immiscible fluid,where the electrodes 608 form a narrowed portion of flow channel 606.(For methods and inlet configurations relevant to flow focusing, see,e.g., US Pub. Nol. 2010/0184928 to Kumacheva.)

Multiplexed embodiments of exemplary FTEP devices are illustrated inFIGS. 7A-7E. FIG. 7A illustrates a top view of a cross section of afirst multiplexed aspect of the FTEP devices of the disclosure. The FTEPdevice in FIG. 7A is a multiplexed FTEP device 700 in which parallelflow channels 706 for each FTEP unit are defined in part by sharedcylindrical electrodes 708 a-708 f forming devices (i), (ii), (iii),(iv), and (v). Each flow channel 706 has an inlet 702 for introducingdifferent sets of cells and/or exogenous materials into the FTEP unitsand an outlet 704 for removing the transformed cells from the FTEPunits. Adjacent units share electrodes, where the electrodes alternatecharge, e.g., +/−/+/−/+(that is, if electrode 708 a is +, electrode 708b is −, electrode 708 c is +, electrode 708 d is −, and so on). FIG. 7Bis an illustration of a top view of a cross section of a secondmultiplexed embodiment of the FTEP devices 710 of the disclosure. Thisis a multiplexed device 710 in which parallel flow channels 706 aredefined in part by shared oval electrodes 708 a-708 f. Each flow channel706 has an inlet 702 for introducing different sets of cells and/orexogenous materials into the flow channels 706, and an outlet forremoving the transformed cells from FTEP units (i), (ii), (iii), (iv),and (v). Again, adjacent devices share electrodes, where the electrodesalternate charge, e.g., +/−/+/−/+.

FIG. 7C is an illustration of a top view of a cross section of a thirdmultiplexed embodiment of the FTEP devices of the disclosure. In thisexemplary multiplexed FTEP device 720, the individual FTEP units arestaggered. The parallel flow channels 706 are defined in part byindividual cylindrical electrodes 708 a-708 j that are not shared asshown in FIGS. 7A and 7B. Each flow channel 706 has its own pair ofelectrodes 708, an inlet 702 for introducing different sets of cellsand/or exogenous materials into the FTEP device, and an outlet forremoving transformed cells from the FTEP units (i), (ii), (iii), (iv),and (v). FIG. 7D is an illustration of a top view of a cross section ofanother exemplary multiplexed FTEP device. In this multiplexed FTEPdevice 730, staggered, parallel flow channels 706 are defined in part byindividual oval electrodes 708 a-708 j. Each flow channel 706 has itsown un-shared pair of electrodes 708 (e.g., 708 a/708 b, 708 c/708 d,708 e/708 f, 708 g/708 h, and 708 i/708 j), an inlet 702 for introducingdifferent sets of cells and/or exogenous materials into the FTEP units,and an outlet 704 for removing transformed cells from the FTEP units.FIG. 7E is an illustration of a top view of a cross section of anotherexemplary multiplexed FTEP device. In this exemplary multiplexed device740, staggered, parallel flow channels 706 are defined in part byindividual half-cylindrical electrodes 708 a-708 j. Each flow channel706 has its own pair of electrodes 708, a separate inlet 702 forintroducing different sets of cells and/or exogenous materials into theFTEP unit, and an outlet 704 for removing the transformed cells from theFTEP unit.

Additional embodiments of the FTEP devices of the disclosure areillustrated in FIGS. 8A-8U. Note that in the FTEP devices in FIGS. 8A-8Uthe electrodes are not positioned on either side of the flow channel tonarrow the flow channel; instead, the electrodes are placed such that afirst electrode is placed between the inlet and the narrowed region ofthe flow channel, and the second electrode is placed between thenarrowed region of the flow channel and the outlet. FIG. 8A shows a topplanar view of an FTEP device 800 having an inlet 802 for introducing afluid containing cells and exogenous material into FTEP device 800 andan outlet 804 for removing the transformed cells from the FTEP followingelectroporation. The electrodes 808 are introduced through channels (notshown) in the device. FIG. 8B shows a cutaway view from the top of theFTEP device 800, with the inlet 802, outlet 804, and electrodes 808positioned with respect to a flow channel 806. FIG. 8C shows a sidecutaway view of FTEP device 800 with the inlet 802 and inlet channel812, and outlet 804 and outlet channel 814. The electrodes 808 arepositioned in electrode channels 816 so that they are in fluidcommunication with the flow channel 806, but not directly in the path ofthe cells traveling through the flow channel 806. Again note that thefirst electrode is placed between the inlet and the narrowed region ofthe flow channel, and the second electrode is placed between thenarrowed region of the flow channel and the outlet.

An expanded side cutaway view of the bottom portion of the device 800 inFIG. 8D shows that the electrodes 808 in this aspect of the device arepositioned in the electrode channels 816 which are generallyperpendicular to the flow channel 806 such that the fluid containing thecells and exogenous material flows from the inlet channel 812 throughthe flow channel 806 to the outlet channel 814, and in the process fluidflows into the electrode channels 816 to be in contact with theelectrodes 808. In this aspect, the inlet channel, outlet channel andelectrode channels all originate from the same planar side of thedevice, as shown in FIGS. 8C and 8D. In certain aspects, however, suchas that shown in FIG. 8E, the electrodes are introduced from a differentplanar side of the FTEP device than the inlet and outlet channels. Here,the electrodes 808 in this alternative aspect of FTEP device 810 arepositioned in the electrode channels 816 perpendicular to the flowchannel 806 such that fluid containing the cells and exogenous materialflow from the inlet channel 812 through the flow channel 806 to theoutlet channel 814. The cells and exogenous material in buffer flow intothe electrode channels 816 to be in contact with both electrodes 808;however, the electrodes 808 are not directly in flow channel 806. Inthis aspect, the inlet channel and outlet channel originate from adifferent planar side of the device than do the electrodes and electrodechannels.

FIGS. 8F-8H illustrate yet another aspect of the FTEP devices of thedisclosure. FIG. 8F shows a top planar view of an FTEP device 820 havinga first inlet 802 for introducing a fluid containing cells into FTEPdevice 820 and an outlet 804 for removing the transformed cells from theFTEP device 820 following electroporation. However, in this FTEP device,there is a second inlet 822 for introducing exogenous material to beelectroporated to the cells. The electrodes 808 are introduced throughchannels (not shown). FIG. 8G shows a cutaway view from the top of theFTEP device 820, with the first inlet 802, second inlet 822, outlet 804,and the electrodes 808 positioned with respect to the flow channel 806.FIG. 8H shows a side cutaway view of FTEP device 820 with inlets 802,822 and inlet channels 812, 824 and outlet 804 and outlet channel 814.The electrodes 808 are positioned in the electrode channels 816 so thatthey are in fluid communication with the flow channel 806, but notsubstantially in the path of the cells traveling through the flowchannel 806. The electrodes 808 in this aspect of the FTEP device 820are positioned in the electrode channels 816 where the electrodechannels 816 are generally perpendicular to the flow channel 806 suchthat fluid containing the cells and fluid containing the exogenousmaterials flow from the inlets 802, 822 through the inlet channels 812,824 into the flow channel 806 and through to the outlet channel 814, andin the process the cells and exogenous material in medium flows into theelectrode channels 816 to be in contact with the electrodes 808. One ofthe two electrodes 808 and electrode channels 816 is positioned betweeninlets 802 and 822 and inlet channels 812 and 824 and the narrowedregion (not shown) of flow channel 806, and the other electrode 808 andelectrode channel 816 is positioned between the narrowed region (notshown) of flow channel 806 and the outlet channel 814 and outlet 804. InFIG. 8H, the inlet channel, outlet channel and electrode channels alloriginate from the same planar side of the device, although theelectrodes (and inlets and outlet) can also be configured to originatefrom a different planar sides of the FTEP device such as illustrated inFIG. 8E.

FIGS. 8I-8M illustrate yet another embodiment of the devices of thedisclosure. FIG. 8I shows a top planar view of an electroporation device830 having an inlet 802 for introducing a fluid containing cells andexogenous material into the FTEP device 830 and an outlet 804 forremoval of the transformed cells from the FTEP device 8300 followingelectroporation. The electrodes 808 are introduced through channels (notshown) machined into the device. FIG. 8J shows a cutaway view from thetop of the device 830, showing an inlet 802, an outlet 804, a filter 850of substantially uniform density, and electrodes 808 positioned withrespect to the flow channel 806. FIG. 8K shows a cutaway view from thetop of an alternative configuration 840 of the device 830, with an inlet802, an outlet 804, a filter 850 of increasing gradient density, andelectrodes 808 positioned with respect to the flow channel 806. In FIGS.8I-8M, like FIGS. 8F-8H, the first electrode is placed between the inletand the narrowed region of the flow channel, and the second electrode isplaced between the narrowed region of the flow channel and the outlet.In some embodiments such as those depicted in FIGS. 8I-8M, the FTEPdevices comprise a filter disposed within the flow channel positioned inthe flow channel after the inlet channel and before the first electrodechannel. The filter may be substantially homogeneous in porosity (e.g.,have a uniform density as in FIG. 8J); alternatively, the filter mayincrease in gradient density where the end of the filter proximal to theinlet is less dense, and the end of the filter proximal to the outlet ismore dense (as shown in FIG. 8K). The filter may be fashioned from anysuitable and preferably inexpensive material, including porous plastics,hydrophobic polyethylene, cotton, glass fibers, or the filter may beintegral with and fabricated as part of the FTEP device body (see, e.g.,FIG. 10E).

FIG. 8L shows a side cutaway view of the device 840 with an inlet 802and an inlet channel 812, and an outlet 804 and an outlet channel 814.The electrodes 808 are positioned in the electrode channels 816 so thatthey are in fluid communication with the flow channel 806, but notdirectly in the path of the cells traveling through flow channel 806.Note that filter 850 is positioned between inlet 802 and inlet channel812 and electrodes 808 and electrode channels 816. An expanded sidecutaway view of the bottom portion of the FTEP device 840 in FIG. 8Mshows that the electrodes 808 in this aspect of the FTEP device 840 arepositioned in the electrode channels 816 and perpendicular to the flowchannel 806 such that fluid containing the cells and exogenous materialflows from the inlet channel 812 through the flow channel 806 to theoutlet channel 814, and in the process fluid flows into the electrodechannels 816 to be in contact with both electrodes 808. In FIGS. 8L and8M, the inlet channel, outlet channel and electrode channels alloriginate from the same planar side of the device, although theelectrodes (and the inlets and outlet) can also be configured tooriginate from a different planar side such as illustrated in FIG. 8E.

FIGS. 8N-8R illustrate other embodiments of the FTEP devices of thedisclosure. FIG. 8N shows a top view of an FTEP device 860 having afirst inlet 802 for introducing a fluid containing cells into the FTEPdevice and a second inlet 818 for introducing a fluid containingexogenous materials to be introduced to the cells into the FTEP device,electrodes 808 positioned in electrode channels (not shown), and anoutlet 804 for removal of the transformed cells followingelectroporation. FIG. 8O shows a cutaway view from the top of the device860, comprising a first inlet 802, second inlet 818, outlet 804, filter850, and electrodes 808 positioned with respect to the flow channel 806.Again note that the electrodes 808 are positioned so that the firstelectrode is on the “inlet end” of the narrowed region in flow channel806 and the second electrode is on the “outlet end” of the narrowedregion in flow channel 806. FIG. 8P shows a first side cutaway view ofan embodiment of the device 860 with the first inlet 802 and secondinlet 818 positioned as shown in FIG. 8N. The first inlet channel 812and second inlet channel 824 meet separately with the flow channel 806prior to encountering filter 850, and the liquid flows from the inletchannels 812 and 824 through the flow channel 806 (and filter 850) tothe outlet channel 814 and outlet 804. Note that in some embodiments,electrodes 808 may be positioned in electrode channels 816 such thatelectrodes 808 are flush with the walls of flow channel 806 (e.g., seeFIG. 10F(iii)). Alternatively, electrodes 808 may extend a minimaldistance into flow channel 806; however, in doing so electrodes 808 donot extend into flow channel 806 to the extent that the electrodesimpede the flow of the cells through the flow channel.

FIG. 8Q shows a side cutaway view of a variation of the embodiment ofthe FTEP device 860 shown in FIGS. 8N-8P with the first inlet 802 andsecond inlet 818 positioned as shown in FIG. 8N. The first inlet channel812 and second inlet channel 824 intersect with flow channel 806 at athree-way junction with flow channel 806 and prior to encounteringfilter 850. The liquid flows through the flow channel 806 to the outletchannel 824 and outlet 804. The electrodes 808 are positioned in theelectrode channels 816 so that they are in fluid communication with theflow channel 806, but not directly in the path of the cells travelingthrough the flow channel 806. Again, the electrodes 808 are positionedso that the first electrode is on the “inlet end” of the narrowed regionin flow channel 806 and the second electrode is on the “outlet end” ofthe narrowed region in flow channel 806. FIG. 8R shows a side cutawayview of yet another variation on the embodiment of the FTEP device 860shown in FIGS. 8N-8P. The first inlet channel 812 and second inletchannel 826 intersect at a junction into a single channel prior tointersecting flow channel 806. The fluids flow from the inlets 802 and818, through the inlet channels 812 and 826, into and through flowchannel 806 and the filter 850, into electrode channels 816 (such thatelectrodes 808 are in fluid communication with flow channel 806) andcontinuing through flow channel 806 to the outlet channel 814 andfinally to the outlet 804 where the transformed cells are removed fromthe FTEP device 860. Again in this embodiment, the electrodes 808 arepositioned in the electrode channels 816 so that they are in fluidcommunication with the flow channel 806, but not directly in the flowpath of the cells traveling through the flow channel 806. Although eachof FIGS. 8P-8R show the inlet channels, outlet channel and electrodechannels originating from the same planar side of the device, all of theinlets, outlet and electrodes in each of these aspects can also beconfigured to originate from different planar sides of the FTEP device.

FIGS. 8S-8U illustrate another embodiment of the FTEP devices of thedisclosure. FIG. 8S shows a top view of an electroporation device 870having a first inlet 802 for introducing a fluid containing cells intoFTEP device 870, a second inlet 818 for introducing exogenous materialsto be porated into the cells into FTEP device 870, and an outlet 804 forremoving transformed cells from FTEP device 870 followingelectroporation. The electrodes 808 are introduced through channels (notshown) machined into the device and are positioned between the firstinlet 802 and the second inlet 818. FIG. 8T shows a cutaway view fromthe top of the device 870, with the first inlet 802, second inlet 818,outlet 804, and the electrodes 808 positioned with respect to the flowchannel 806. Additionally, the FTEP device depicted in FIG. 8T comprisesa filter 850 disposed between the first inlet 802 and the firstelectrode 808 and before the narrowed region of flow channel 806. Filter850 in this embodiment has a gradient of pore sizes, from large to small(moving from the inlet 802 toward the narrowed portion of flow channel806. FIG. 8U shows a side cutaway view of FTEP device 870 comprising afirst inlet 802 and first inlet channel 812, a filter 850, a secondinlet 818 and second inlet channel 832, and an outlet 804 and outletchannel 814. The electrodes 808 are positioned in the electrode channels816 perpendicular to flow channel 806 and between the first and secondinlets. The electrodes 808 are in fluid communication with flow channel806, but not in the flow channel and thus in the path of the cellstraveling through flow channel 806. Exogenous materials are added toFTEP device 870 via the second inlet 818 and through the second inletchannel 832 and encounter the cells after the cells are electroporated.In FIG. 8U, the inlet channels, outlet channel and electrode channelsall originate from the same planar side of the device, although thesefeatures can also be configured to originate from different planar sidesof FTEP device 870.

FIGS. 9A and 9B show the side and top cutaway views, respectively, ofyet another embodiment of the invention. FIG. 9A shows a multilayerdevice 900 with a top layer 952 having an inlet 902 and an inlet channel912, a flow channel 906, and outlet 904 and an outlet channel 914. Theelectrodes 908 are on bottom layer 956, e.g., provided as strips on asolid substrate. The middle layer 954 is a solid substrate withelectrode channels 916 provided therein, and the electrode channels 916in this aspect provide fluid communication between the electrodes 908 ofbottom layer 956 and flow channel 906 of top layer 952. The cells andexogenous materials in fluid are introduced to the FTEP device 900 viainlet 902 and flow through inlet channel 912 and into flow channel 906,and then to the outlet channel 914. In the process, the fluid flows intoelectrode channels 916 so that electrodes 908 are in fluid contact withflow channel 906. The cells are porated as they pass through flowchannel 906 between the two electrodes 908. FIG. 9B shows the top viewof a cutaway 910 of the embodiment of the FTEP device 900 showing theposition of the inlet 902, outlet 904, electrodes 908 and electrodechannels 916 with respect to the flow channel 906. Although theelectrodes are shown here as strips, they may also be configured to beother shapes, e.g., round, cylindrical, asymmetric, rectangular, orsquare.

FIG. 9C illustrates an FTEP device in which flow focusing 930 of thefluid introduced into the flow channel from the input channel(s) takesplace, e.g., using an immiscible fluid such as an oil or using air tofocus (narrow) the stream of the fluid containing the cells andexogenous materials as the fluid encounters the electrode channels, andthe electrodes. FIG. 9C shows a cutaway view from the top of the device920, with the first inlet 902, the flow focusing 930 of the fluid afterit exits the inlet channel and enters the flow channel 906, and theelectrodes 908 positioned between the inlet 902 and the outlet 904,where the electrodes 908 are positioned on either end of a narrowedportion of flow channel 906.

The reagent cartridges for use in the automated multi-module cellediting instruments (e.g., cartridge 104 of FIG. 1A), in someembodiments, include one or more FTEP devices (e.g., electroporationmodule 110 c of FIG. 1A, also see 1124 of FIG. 11E).

FIGS. 10A through 10C are top perspective, bottom perspective, andbottom views, respectively, of six co-joined FTEP devices 1050 that maybe part of, e.g., reagent cartridge 1122 in FIG. 11E infra (i.e., serveas FTEP 1124 in reagent cartridge 1122). FIG. 10A depicts six FTEP units1050 (i.e., (i), (ii), (iii), (iv), (v), and (vi)) arranged on a single,integrally-formed injection molded cyclic olefin copolymer (COC)substrate 1056. The channels 1006 shown in FIG. 10B are sealed with aCOC film having a thickness of 50 microns to 1 mm (not shown). The COCfilm may be thermally bonded to the base of the assembly 1000 (thesurface most prominently displayed in FIG. 10B). In FIGS. 10B and 10C,the co-joined FTEP devices have different channel architectures andelectrode placements that may be advantageous in various applications.For instance, the curved channels of devices (i), (iv) and (v) takeadvantage of inertia to direct the cells in the fluid away from theelectrodes. The electrodes may be positioned off center in the channelto further enhance cells flow and reduce the potential for damage to thecells. This may be particularly important for cells or materials thatare particularly sensitive to electrolytic effects or local changes inpH proximate the electrodes. The electrodes may be at least partiallyembedded into the channel walls, as shown in embodiments (iii) and (iv),so as to further reduce these effects.

Each of the six FTEP units 1050 have wells or reservoirs 1052 thatdefine cell sample inlets and wells 1054 that define cell sampleoutlets. FIG. 10B is a bottom perspective view of the six co-joined FTEPdevices 1050 of FIG. 15A also depicting six FTEP units 1050 (i.e.,(i)-(vi)) arranged on a single substrate 1056. Six inlet wells 1052 canbe seen, one for each flow-through electroporation unit 1050, and oneoutlet well 1054 can be seen. Also seen in FIG. 10B for each FTEP unit1050 are an inlet 1002, an outlet 1004, a flow channel 1006, and twoelectrodes 1008 on either end of a narrowed region in flow channel 1006.Filters 1070 and 1072 are included in the channels to prevent cloggingof the channel, particularly at narrowed region of the flow channel.FIG. 10C is a bottom view of the six co-joined FTEP devices 1050 ofFIGS. 10A and 10B. Depicted in FIG. 10C are six FTEP units 1050 (i.e.,(i)-(vi)) arranged on a single substrate 1056, where each FTEP unit 1050comprises an inlet 1002, outlet 1004, flow channel 1006 and twoelectrodes 1508 on either end of a narrowed region in flow channel 1006in each FTEP unit 1050. Once the six FTEP units 1050 are fabricated,they can be separated from one another (e.g., “snapped apart”) upon thedepicted score lines and used one at a time as seen in the cartridgedepicted in FIG. 11E; alternatively, the FTEP units may be used inembodiments where two or more FTEP units 1050 are used in parallel.

FIG. 10D shows scanning electromicrographs of the FTEP units depicted inFIG. 10C with the units (i), (ii), (iii), (iv), (v), and (vi) in FIG.10D corresponding to units (i), (ii), (iii), (iv), (v), and (vi) in FIG.10C. In FIG. 10D, for each unit both the electrode channels 1016 as wellas the flow channel 1006 can be seen.

FIG. 10E shows scanning electromicrographs of the filters 1070 and 1072depicted as black bars in FIGS. 10B and 10C. Note in this embodiment,the porosity of the filter 1072 varies from large pores (near the inlet1002) to small pores toward the flow channel (not shown). In thisembodiment, the channel optionally but not necessarily narrows. If asecond filter is present, the second filter may also vary in porosity.In the case of a second filter between the second electrode and theoutlet channel, the filter can vary from large pores (near the secondelectrode) to small pores toward the outlet channel. Scale informationis shown in each micrograph.

In certain embodiments, the filter serves the purpose of filtering thefluid containing the cells and DNA before the fluid encounters thenarrowed portion of the flow channel. The filter thus decreases thelikelihood that cells or other matter do not clog the narrowed portionof the flow channel. Instead, if there is particulate matter that posesa threat to clogging the narrowed portion of the flow channel, thefilter will catch the particulate matter leaving other pores throughwhich the rest of the cell/DNA/fluid can move. The depicted construction(integral molding with the channel wall) is particularly advantageousbecause it reduces cost and complexity of the device while also reducingthe risk that pieces of the filter itself may dislodge and clog thechannel or otherwise interfere with device operation. Note that in thisembodiment, the filter has a gradient pore size (from large poresproximate the inlet to smaller pores proximal the narrowed portion ofthe flow channel); however, in alternative embodiments the pores may bethe same size or not gradient in size.

Further, in yet other embodiments the flow channel may not narrow. Inthese specific embodiments, the pores themselves can serve to providesuch a narrowing function for enhancing electroporation, and the flowchannel walls do not narrow or narrow minimally as the fluid flowsthrough the channel. These embodiments can allow control of the rate offlow of cells through the device to optimize introduction of exogenousmaterial into various cell types.

Moreover, though the scanning electromicrographs in FIG. 10E shown thefilter elements as rounded “pegs”, it should be understood that thefilter elements may be triangular-, square-, rectangular-, pentagonal-,hexagonal-, oval-, elliptical- or other faceted-shaped pegs.

FIG. 10F depicts (i) the electrodes 1008 before insertion into the FTEPdevice 1000 (here, a six-unit FTEP device) having inlet reservoirs 1052and outlet reservoirs 1054. In the preferred embodiment, the device 1000is used in an orientation inverted relative to that shown in FIG. 10F(i). FIG. 10F (ii) depicts an electrode 1008 contained within andprojecting from a sheath. FIG. 10F (iii) depicts the electrode 1008inserted into an electrode channel 1016 with the electrode channel 1016(and electrode 1008) adjacent to the flow channel 1006. In theembodiment shown in FIG. 10F (iii), the electrode is even with the wallsof the flow channel; that is, the electrode is not in the path of thecells/DNA/fluid flowing through flow channel 1006, however, neither isthe electrode recessed within the electrode channel 1016. Indeed, theelectrode 1008 may be recessed within the electrode channel 1016, may beextend to the end of electrode channel 1016 and thus be even with thewalls of flow channel 1006, or electrode 1008 may extend a minimaldistance into flow channel 1006 so long as the electrode does not impedemovement of the cells through the flow channel. The rounded or bevelededges of the aperture in the flow channel 1006 help prevent trapping airand reduce discontinuities in the electric field.

FIG. 10G presents two scanning electromicrographs of two differentconfigurations of the aperture where electrode channel 1016 meets flowchannel 1006. In FIG. 10G (i) (top), the edge of the junction ofelectrode channel 1016 and flow channel 1506 comprises a sharp edge. Incontrast, in FIG. 10G (ii) (bottom), the edges of the junction ofelectrode channel 1016 and flow channel 1006 comprises a rounded edge.Both configurations were tested (data not shown), and it was found thatthe rounded-edge configuration decreases the likelihood that air willbecome trapped between flow channel 1006 and the electrode (not seen inthis Figure) in electrode channel 1016. It can be seen that in thisembodiment the inlet apertures have a rounded edge, the advantages ofwhich include resistance to air trapping, promotion of laminar flow, andreduction of risk of cell damage. The rounded edges may have a radius ofcurvature of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 or250 microns. Indeed, the electrodes of the FTEP devices should be “wet”;that is, immersed in the fluid/cells/DNA.

After transformation, the cells are allowed to recover under conditionsthat promote the genome editing process that takes place as a result ofthe transformation and expression of the introduced nucleic acids in thecells.

Methods for Automated Multi-module Cell Editing

FIG. 14 is a flow chart of an example method 1400 for using an automatedmulti-module cell editing instrument such as the systems illustrated inFIGS. 1A-1B and 17A-17B. Further, the processing system of FIG. 18, forexample, may direct the processing stage of the method 1400. Forexample, a software script may identify settings for each processingstage and instructions for movement of a robotic handling system toperform the actions of the method 1400. In some embodiments, a softwareinstruction script may be identified by a cartridge supplied to theautomated multi-module cell editing instrument. For example, thecartridge may include machine-readable indicia, such as a bar code or QRcode, including identification of a script stored in a memory of theautomated multi-module cell editing instrument (e.g., such as memory1802 of FIG. 18). In another example, the cartridge may contain adownloadable script embedded in machine-readable indicia such as a radiofrequency (RF) tag. In other embodiments, the user may identify ascript, for example through downloading the script via a wired orwireless connection to the processing system of the automatedmulti-module cell editing instrument or through selecting a storedscript through a user interface of the automated multi-module cellediting instrument. In a particular example, the automated multi-modulecell editing instrument may include a touch screen interface forsubmitting user settings and activating cell processing.

In some implementations, the method 1400 begins with transferring cellsto a growth module (1402). The growth module may be any growth moduleamendable to automation, for example, may be the growth module 1350described in relation to FIGS. 13B and 13C. In a particular example, theprocessing system 1720 may direct the robotic handling system 1708 totransfer cells 1706 to the growth module 1710 a, as described inrelation to FIGS. 17A and 17B. In another example, as described inrelation to FIG. 1A, the cells may be transferred from one of thecartridges 104, 106 to the growth modules 110 a, 110 b by the robotichandling system 108. In some embodiments, the growth vial may containgrowth media and be supplied, e.g., as part of a kit. In otherembodiments, the growth vial may be filled with medium transferred,e.g., via the liquid handling device, from a reagent container.

In some embodiments, prior to transferring the cells (e.g., from thereagent cartridge or from a vial added to the instrument),machine-readable indicia may be scanned upon the vial or other containersituated in a position designated for cells to confirm that the vial orcontainer is marked as containing cells. Further, the machine-readableindicia may indicate a type of cells provided to the instrument. Thetype of cells, in some embodiments, may cause the instrument to select aparticular processing script (e.g., series of instructions for therobotic handling system and settings and activation of the variousmodules).

In some implementations, the cells are grown in the growth module to adesired optical density (1404). For example, the processing system 126of FIGS. 1A-1B or processing system 1720 of FIGS. 17A-B may manage atemperature setting of the growth module 110 a for incubating the cellsduring the growth cycle. The processing system 126, 1720 may furtherreceive sensor signals from the growth module 110 a, 110 b, 1710 aindicative of optical density and analyze the sensor signals to monitorgrowth of the cells. In some embodiments, a user may set growthparameters for managing growth of the cells. For example, temperature,and the degree of agitation of the cells. Further, in some embodiments,the user may be updated regarding growth process. The updates, in someexamples, may include a message presented on a user interface of theautomated multi-module cell editing instrument, a text message to auser's cell phone number, an email message to an email account, or amessage transmitted to an app executing upon a portable electronicdevice (e.g., cell phone, tablet, etc.). Responsive to the messages, insome embodiments, the user may modify parameters, such as temperature,to adjust cell growth. For example, the user may submit updatedparameters through a user interface of the automated multi-module cellediting instrument or through a portable computing device application incommunication with the automated multi-module cell editing instrument,such as a user interface 1600 of FIG. 16.

Although described in relation to optical density, in otherimplementations cell growth within the growth module may be monitoredusing a different measure of cell density and physiological state suchas, in some examples, pH, dissolved oxygen, released enzymes, acousticproperties, and electrical properties.

In some implementations, upon reaching the desired optical density(1404), the cells are transferred from the growth module to a filtrationmodule or cell wash and concentration module (1406). The robotichandling system 108 of FIGS. 1A-1B or 1708 of FIGS. 17A-17B, forexample, may transfer the cells from the growth module 1710 a to thefiltration module 1710 b. The filtration module, for example, may bedesigned to render the cells electrocompetent. Further, the filtrationmodule may be configured to reduce the volume of the cell sample to avolume appropriate for electroporation. In another example, thefiltration module may be configured to remove unwanted components, suchas salts, from the cell sample. In some examples, the filtration systemperforms two of the preceding tasks, and in certain preferredembodiments all three of the preceding tasks, rendering the cellselectrocompetent, reducing the volume of the fluid containing the cells,and removing unwanted components. In some embodiments, the robotichandling system 1708 transfers a washing solution to the filtrationmodule 1710 b for washing the cells.

In some implementations, the cells are rendered electrocompetent andeluted in the filtration module or cell wash and concentration module(1408). The cells may be eluted using a wash solution. For example, thecells may be eluted using reagents from a reagent supply. The filtrationmodule or cell wash and concentration module, for example, may besimilar to the filtration module 1200 described in relation to in FIGS.12A-12D. As discussed above, numerous different methods can be used towash the cells, including density gradient purification, dialysis, ionexchange columns, filtration, centrifugation, dilution, and the use ofbeads for purification. In some aspects, the cell wash and concentrationmodule utilizes a centrifugation device. In other aspects, thefiltration module utilizes a filtration instrument. In yet otheraspects, the beads are coupled to moieties that bind to the cellsurface. These moieties include but are not limited to antibodies,lectins, wheat germ agglutinin, mutated lysozymes, and ligands. In otheraspects, the cells are engineered to be magnetized, allowing magnets topellet the cells after wash steps. Mechanism of cell magnetization caninclude but not limited to ferritin protein expression.

In some embodiments, the wash solution is transferred to the filtrationmodule prior to eluting. The robotic handling system 1708 of FIGS.17A-17B, for example, may transfer the wash solution from a vial orcontainer situated in a position designated for wash solution. Prior totransferring the wash solution, machine-readable indicia may be scannedupon the vial or other container or reservoir situated in the positiondesignated for the wash solution to confirm the contents of the vial,container, or reservoir. Further, the machine-readable indicia mayindicate a type of wash solution provided to the instrument. The type ofwash solution, in some embodiments, may cause the system to select aparticular processing script (e.g., settings and activation of thefiltration module appropriate for the particular wash solution).

In other embodiments, the cells are eluted in a cell wash module of awash cartridge. For example, the eluted cells may be collected in anempty vessel of the wash cartridge 106 illustrated in FIG. 1A, and therobotic handling system 108 may transfer media from the reagentcartridge 104 (or a reagent well of the wash cartridge 106) to theeluted cells.

Once the cells have been rendered electrocompetent and suspended in anappropriate volume such as 50 μL to 10 mL, or 100 μL to 80 mL, or 150 μLto 8 mL, or 250 μL to 7 mL, or 500 μL to 6 mL, or 750 μL to 5 mL fortransformation by the filtration module (1406), in some implementations,the cells are transferred to an FTEP module (1418). The robotic handlingsystem 108 of FIGS. 1A-1B, for example, may transfer the cells from thefiltration module to the FTEP device 110 c. The filtration module may bephysically coupled to the FTEP device, or these modules may be separate.In an embodiment such as the instrument 100 of FIG. 1A havingcartridge-based supplies, the cells may be eluted to a reservoir withina cartridge, such as the reagent cartridge 104, prior to transferring tothe transformation module.

In some implementations, nucleic acids are prepared outside of theautomated multi-module cell editing instrument. For example, anassembled vector or other nucleic acid assembly may be included as areagent by a user prior to running the transformation process and otherprocesses in the method 1400.

However, in other implementations, nucleic acids are prepared by theautomated multi-module cell editing instrument. A portion of thefollowing steps 1410 through 1416, in some embodiments, are performed inparallel with a portion of steps 1402 through 1408. At least a portionof the following steps, in some embodiments, are performed before and/orafter steps 1402 through 1408.

In some implementations nucleic acids such as an editing oligonucleotideand a vector backbone, as well as, in some examples, enzymes and otherreaction components are transferred to a nucleic acid assembly module(1410). The nucleic acid assembly module may be configured to performone or more of a wide variety of different nucleic acid assemblytechniques in an automated fashion. Nucleic acid assembly techniquesthat can be performed in the nucleic acid assembly module may include,but are not limited to, those assembly methods that use restrictionendonucleases, including PCR, BioBrick assembly, Type IIS cloning,GoldenGate assembly, and Ligase Cycling Reaction. In other examples, thenucleic acid assembly module may perform an assembly technique based onoverlaps between adjacent parts of the nucleic acids, such as GibsonAssembly®, CPEC, SLIC, Ligase Cycling etc. Additional example assemblymethods that may be performed by the nucleic acid assembly moduleinclude gap repair in yeast, gateway cloning and topoisomerase-mediatedcloning. The nucleic acid assembly module, for example, may be thenucleic acid assembly module 300 described in relation to FIG. 3. In aparticular example, the processing system 1720 may direct the robotichandling system 1708 to transfer nucleic acids 1704 to the nucleic acidassembly module 1710 g, as described in relation to FIG. 17B. In anotherexample, as described in relation to FIG. 1A, the nucleic acids may betransferred from one of the cartridges 104, 106 to a nucleic acidassembly module by the robotic handling system 108.

In some embodiments—prior to transferring each of the nucleic acidsamples, the enzymes, and other reaction components—machine-readableindicia may be scanned upon the vials or other containers situated inpositions designated for these materials to confirm that the vials orcontainers are marked as containing the anticipated material. Further,the machine-readable indicia may indicate a type of one or more of thenucleic acid samples, the enzymes, and other reaction componentsprovided to the instrument. The type(s) of materials, in someembodiments, may cause the instrument to select a particular processingscript (e.g., series of instructions for the robotic handling system toidentify further materials and/or settings and activation of the nucleicacid assembly module).

In some embodiments, the nucleic acid assembly module is temperaturecontrolled depending upon the type of nucleic acid assembly used. Forexample, when PCR is utilized in the nucleic acid assembly module, themodule can have a thermocycling capability allowing the temperatures tocycle between denaturation, annealing and extension. When singletemperature assembly methods are utilized in the nucleic acid assemblymodule, the module can have the ability to reach and hold at thetemperature that optimizes the specific assembly process beingperformed.

Temperature control, in some embodiments, is managed by a processingsystem of the automated multi-module cell editing instrument, such asthe processing system 1810 of FIG. 18. These temperatures and theduration of maintaining the temperatures can be determined by apreprogrammed set of parameters (e.g., identified within the processingscript or in another memory space accessible by the processing system),or manually controlled by the user through interfacing with theprocessing system.

Once sufficient time has elapsed for the assembly reaction to takeplace, in some implementations, the nucleic acid assembly is transferredto a purification module (1414). The processing system, for example, maymonitor timing of the assembly reaction based upon one or more of thetype of reaction, the type of materials, and user settings provided tothe automated multi-module cell editing instrument. The robotic handlingsystem 108 of FIGS. 1A-1B or 1708 of FIGS. 17A-17B, for example, maytransfer the nucleic acid assembly to the purification module through asipper or pipettor interface. In another example, the robotic handlingsystem 108 of FIGS. 1A-1B or 1708 of FIGS. 17A-17B may transfer a vialcontaining the nucleic acid assembly from a chamber of the nucleic acidassembly module to a chamber of the de-salt/purification module.

In some implementations, the nucleic acid assembly is de-salted andeluted at the purification module (1416). The purification module, forexample, may remove unwanted components of the nucleic acid assemblymixture (e.g., salts, minerals, etc.). In some embodiments, thepurification module concentrates the assembled nucleic acids into asmaller volume that the nucleic acid assembly volume. Examples ofmethods for exchanging liquid following nucleic acid assembly includemagnetic beads (e.g., SPRI or Dynal (Dynabeads) by Invitrogen Corp. ofCarlsbad, Calif.), silica beads, silica spin columns, glass beads,precipitation (e.g., using ethanol or isopropanol), alkaline lysis,osmotic purification, extraction with butanol, membrane-based separationtechniques, filtration etc. For example, one or more micro-concentratorsfitted with anisotropic, hydrophilic-generated cellulose membranes ofvarying porosities may be used. In another example, thede-salt/purification module may process a liquid sample including anucleic acid and an ionic salt by contacting the mixture with an ionexchanger including an insoluble phosphate salt, removing the liquid,and eluting nucleic acid from the ion exchanger.

In an illustrative embodiment, the nucleic acid assembly may be combinedwith magnetic beads, such as SPRI beads, in a chamber of a purificationmodule. The nucleic acid assembly may be incubated at a set temperaturefor sufficient time for the assembled nucleic acids to bind to themagnetic beads. After incubation, a magnet may be engaged proximate tothe chamber so that the nucleic acid assembly can be washed and eluted.An illustrative example of this process is discussed in relation to thecombination nucleic acid assembly and purification module of FIG. 3.

Once the nucleic acid assembly has been eluted, the nucleic acidassembly, in some implementations, is transferred to the transformationmodule (1418). The robotic handling system 108 of FIGS. 1A-1B or 1708 ofFIGS. 17A-17B, for example, may transfer the assembled nucleic acids tothe transformation module through a sipper or pipettor interface to theFTEP as described above. For example, the de-salted assembled nucleicacids, during the transfer, may be combined with the electrocompetentcells from step 1408. In other embodiments, the transformation modulemay accept each of the electrocompetent cells and the nucleic acidassembly separately and enable the mixing (e.g., open one or morechannels to combine the materials in a shared chamber).

The cells are transformed in the FTEP module (1420). A buffer or mediummay be transferred to the transformation module and added to the cellsso that the cells may be suspended in a buffer or medium that isfavorable for cell survival during electroporation. Prior totransferring the buffer or medium, machine-readable indicia may bescanned upon the vial or other container or reservoir situated in theposition designated for the buffer or medium to confirm the contents ofthe vial, container, or reservoir. Further, the machine-readable indiciamay indicate a type of buffer or medium provided to the instrument. Thetype of buffer or medium, in some embodiments, may cause the instrumentto select a particular processing script (e.g., settings and activationof the transformation module appropriate for the particular buffer ormedium). For bacterial cell electroporation, low conductance mediums,such as water or glycerol solutions, may be used to reduce the heatproduction by transient high current. For yeast cells a sorbitolsolution may be used. For mammalian cell electroporation, cells may besuspended in a highly conductive medium or buffer, such as MEM, DMEM,IMDM, RPMI, Hanks', PBS, HBSS, HeBS and Ringer's solution. In aparticular example, the robotic handling system 108 (FIG. 1A) maytransfer a buffer solution to FTEP module 110 c from one of thecartridges 104, 106. As described in relation to FIGS. 4A-4I, 5A-5H, 6,7A-7E, 8A-8U, and 9A-9C, the FTEP device may be a disposable FTEP deviceand/or the FTEP device 110 c may be provided with the cartridge 104 ofFIG. 1A (or FTEP device 1124 of cartridge 1122 in FIG. 11E).

Once transformed, the cells are transferred to a secondgrowth/recovery/editing module (1422). The robotic handling system 108of FIGS. 1A-1B or 1708 of FIGS. 17A-17B, for example, may transfer thetransformed cells to the second growth module through a sipper orpipettor interface. In another example, the robotic handling system 108of 1A-1B or 1708 of FIGS. 17A-17B may transfer a vial containing thetransformed cells from a chamber of the transformation module to achamber of the second growth module.

The second growth module, in some embodiments, acts as a recoverymodule, allowing the cells to recover from the transformation process.In other embodiments, the cells may be provided to a separate recoverymodule prior to being transported to the second growth module. Duringrecovery, the second growth module allows the transformed cells touptake and, in certain aspects, integrate the introduced nucleic acidsinto the genome of the cell. The second growth module may be configuredto incubate the cells at any user-defined temperature optimal for cellgrowth, preferably 25°, 30°, or 37° C.

In some embodiments, the second growth module behaves as a selectionmodule, selecting the transformed cells based on an antibiotic or otherreagent. In one example, the RNA-guided nuclease (RGN) protein system isused for selection to cleave the genomes of cells that have not receivedthe desired edit. The RGN protein system used for selection can eitherbe the same or different as the RGN used for editing. In the example ofan antibiotic selection agent, the antibiotic may be added to the secondgrowth module to enact selection. Suitable antibiotic resistance genesinclude, but are not limited to, genes such as ampicillin-resistancegene, tetracycline-resistance gene, kanamycin-resistance gene,neomycin-resistance gene, canavanine-resistance gene,blasticidin-resistance gene, hygromycin-resistance gene,puromycin-resistance gene, or chloramphenicol-resistance gene. Therobotic handling system 108 of FIGS. 1A-1B or 1708 of FIGS. 17A-17B, forexample, may transfer the antibiotic to the second growth module througha sipper or pipettor interface. In some embodiments, removing dead cellbackground is aided using lytic enhancers such as detergents, osmoticstress by hyponic wash, temperature, enzymes, proteases, bacteriophage,reducing agents, or chaotropes. The processing system 1810 of FIG. 18,for example, may alter environmental variables, such as temperature, toinduce selection, while the robotic handling system 108 of FIGS. 1A-1Bor 1708 of FIGS. 17A-17B may deliver additional materials (e.g.,detergents, enzymes, reducing agents, etc.) to aid in selection. Inother embodiments, cell removal and/or media exchange by filtration isused to reduce dead cell background.

In further embodiments, in addition to or as an alternative to applyingselection, the second growth module serves as an editing module,allowing for genome editing in the transformed cells. Alternatively, inother embodiments the cells post-recovery and selection (if performed)are transferred to a separate editing module. As an editing module, thesecond growth module induces editing of the cells' genomes, e.g.,through facilitating expression of the introduced nucleic acids.Expression of the nuclease and/or editing cassette nucleic acids mayinvolve one or more of chemical, light, viral, or temperature inductionmethods. The second growth module, for example, may be configured toheat or cool the cells during a temperature induction process. In aparticular illustration, the cells may be induced by heating at 42°C.-50° C. Further to the illustration, the cells may then be are cooledto 0-10° C. after induction. In the example of chemical or viralinduction, an inducing agent may be transferred to the second growthmodule to induce editing. If an inducible nuclease and/or editingcassette was introduced to the cells during editing, it can be inducedthrough introduction of an inducer molecule, such as the inducermolecule 1724 described in relation to FIG. 17A. The inducing agent orinducer molecule, in some implementations, is transferred to the secondgrowth module by the robotic handling system 108 of FIGS. 1A-1B or 1708of FIGS. 17A-17B (e.g., through a pipettor or sipper interface).

In some implementations, if no additional cell editing is desired(1424), the cells may be transferred from the cell growth module to astorage unit for later removal from the automated multi-module cellediting instrument (1426). The storage unit, for example, may includethe storage unit 1714 of FIGS. 17A-17B. The robotic handling system 108of FIGS. 1A-1B or 1708 of FIGS. 17A-17B, for example, may transfer thecells to the storage unit 114 through a sipper or pipettor interface. Inanother example, the robotic handling system 108 of FIGS. 1A-1B or 1708of FIGS. 17A-17B may transfer a vial containing the cells from a chamberof the second growth module to a vial or tube within the storage unit.

In some implementations, if additional cell editing is desired (1424),the cells may be transferred to the same or a different filtrationmodule and rendered electrocompetent (1408). Further, in someembodiments, a new assembled nucleic acid sample may be prepared by thenucleic acid assembly module at this time, or, alternatively, a secondfully assembled nucleic acid may be directly introduced to the cells.Prior to recursive editing, in some embodiments, the automatedmulti-module cell editing instrument may require additional materials besupplied by the user, e.g., through the introduction of one or moreseparate reagents vails or cartridge.

The steps may be the same or different during the second round ofediting. For example, in some embodiments, upon a subsequent executionof step 1404, a selective growth medium is transferred to the growthmodule to enable selection of edited cells from the first round ofediting. The robotic handling system 108 of FIGS. 1A-B or 1708 of FIGS.17A-17B, for example, may transfer the selective growth medium from avial or container in a reagent cartridge situated in a positiondesignated for selective growth medium. Prior to transferring theselective growth medium, machine-readable indicia may be scanned uponthe vial or other container or reservoir situated in the positiondesignated for the selective growth medium to confirm the contents ofthe vial, container, or reservoir. Further, the machine-readable indiciamay indicate a type of selective growth medium provided to theinstrument. The type of selective growth medium, in some embodiments,may cause the instrument to select a particular processing script (e.g.,settings and activation of the growth module appropriate for theparticular selective growth medium). Particular examples of recursiveediting workflows are described in relation to FIGS. 15A through 15C.

In some implementations, the method 1400 can be timed to introducematerials and/or complete the editing cycle or growth cycle incoordination with a user's schedule. For example, the automatedmulti-module cell editing instrument may provide the user the ability toschedule completion of one or more cell processing cycles (e.g., one ormore recursive edits) such that the method 1400 is enacted with a goalof completion at the user's preferred time. The time scheduling, forexample, may be set through a user interface, such as the user interface1816 of FIG. 18. For illustration only, a user may set completion of afirst cycle to 4:00 PM so that the user can supply additional cartridgesof materials to the automated multi-module cell editing instrument toenable overnight processing of another round of cell editing. Thus auser may time the programs so that two or more cycles may be programmedin a specific time period, e.g., a 24-hour period.

In some implementations, throughout the method 1400, the automatedmulti-module cell editing instrument may alert the user to its currentstatus. For example, the user interface 1816 of FIG. 18 may present agraphical indication of the present stage of processing. In a particularexample, a front face of the automated multi-module call processinginstrument may be overlaid with a user interface (e.g., touch screen)that presents an animated graphic depicting present status of the cellprocessing. The user interface may further present any user and/ordefault settings associated with the current processing stage (e.g.,temperature setting, time setting, etc.). In certain implementations,the status may be communicated to a user via wireless communicationscontroller.

Although illustrated as a particular series of operations, in otherembodiments, more or fewer steps may be included in the method 1400. Forexample, in some embodiments, prior to engaging in each round ofediting, the contents of reservoirs, cartridges, and/or vials may bescreened to confirm appropriate materials are available to proceed withprocessing. For example, in some embodiments, one or more imagingsensors (e.g., barcode scanners, cameras, etc.) may confirm contents atvarious locations within the housing of the automated multi-module cellediting instrument. In one example, multiple imaging sensors may bedisposed within the housing of the automated multi-module cell editinginstrument, each imaging sensor configured to detect one or morematerials (e.g., machine-readable indicia such as barcodes or QR codes,shapes/sizes of materials, etc.). In another example, at least oneimaging sensor may be moved by the robotic handling system to multiplelocations to detect one or more materials. In further embodiments, oneor more weight sensors may detect presence or absence of disposable orreplaceable materials. In an illustrative example, the transfer tipsupply holder may include a weight sensor to detect whether or not tipshave been loaded into the region. In another illustrative example, anoptical sensor may detect that a level of liquid waste has reached athreshold level, requiring disposal prior to continuation of cellprocessing or addition of liquid if the minimum level has not beenreached to proceed. Requests for additional materials, removal of wastesupplies, or other user interventions (e.g., manual cleaning of one ormore elements, etc.), in some implementations, are presented on agraphical user interface of the automated multi-module cell editinginstrument. The automated multi-module cell editing instrument, in someimplementations, contacts the user with requests for new materials orother manual interventions, for example through a software app, email,or text message.

Workflows for Cell Processing in an Automated Multi-module Cell EditingInstrument

The automated multi-module cell editing instrument is designed toperform a variety of cell processing workflows using the same modules.For example, source materials, in individual containers or in cartridgeform, may differ and the corresponding instructions (e.g., softwarescript) may be selected accordingly, using the same basicinstrumentation and robotic handling system; that is, the multi-modulecell editing instrument can be configured to perform a number ofdifferent workflows for processing cell samples and different types ofcell samples. In embodiments, a same workflow may be performediteratively to recursively edit a cell sample. In other embodiments, acell sample is recursively edited, but the workflow may change fromiteration to iteration.

FIGS. 15A through 15C illustrate example workflows that may be performedusing an automated multi-module cell editing instrument including twocell growth modules 1502, 1508, two filtration modules 1504 and 1510,and a flow-through electroporation module 1506. Although described asseparate growth modules 1502, 1508 and filtration modules 1504, 1510,each may instead be designed as a dual or integrated module. Forexample, a dual growth module, including growth modules 1502 and 1508,may include dual rotating growth vials sharing some circuitry, controls,and a power source and disposed in a same housing. Similarly, a dualfiltration module may include filtration modules 1504 and 1510,including two separate filters and liquid supply tubes but sharingcircuitry, controls, a power source, and a housing. The modules 1502,1504, 1506, 1508, and 1510, for example, may be part of the instrument100 described in relation to FIGS. 1A and 1B.

Turning to FIG. 15A, a flow diagram illustrates a first bacteria genomeediting workflow 1500 involving two stages of processing havingidentical processing steps, resulting in two edits to a cell stock 1512.Each stage may operate based upon a different cartridge of sourcematerials. For example, a first cartridge may include a first oligolibrary 1514 a and a first vector backbone, e.g., an expression plasmid1516 a. A second cartridge, introduced into the automated multi-modulecell editing instrument between processing stages or prior to processingbut in a different position than the first cartridge, may include asecond oligo library 1514 b and a second vector backbone 1516 b. Eachcartridge may be considered as a “library cartridge” for building alibrary of edited cells. The cell stock 1512, in some embodiments, isincluded in the first library cartridge. The cell stock 1512 may besupplied within a kit including the two cartridges. Alternatively, auser may add a container (e.g., vial or tube) of the cell stock 1512 toa purchased cartridge.

The workflow 1500, in some embodiments, is performed based upon a scriptexecuted by a processing system of the automated multi-module cellediting instrument, such as the processing system 1810 of FIG. 18. Thescript, in a first example, may be accessed via a machine-readablemarker or tag added to the first cartridge. In some embodiments, eachprocessing stage is performed using a separate script. For example, eachcartridge may include an indication of a script or a script itself forprocessing the contents of the cartridge.

In some implementations, the first stage begins with introducing thecell stock 1512 into the first growth module 1502 for inoculation,growth, and monitoring (1518 a). In one example, a robotic handlingsystem adds a vial of the cell stock 1512 to medium contained in therotating growth vial of the first growth module 1502. In anotherexample, the robotic handling system pipettes cell stock 1512 from thefirst cartridge and adds the cell stock 1012 to the medium contained inthe rotating growth vial. The cells may have been maintained at atemperature of 4° C. at this point. In a particular example, 20 ml ofcell stock may be grown within a rotating growth vial of the firstgrowth module 1002 at a temperature of 30° C. to an OD of 0.50. The cellstock 1012 added to the first growth module 1502 may be monitored overtime until 0.50 OD is sensed via automated monitoring of the growthvial. Monitoring may be periodic or continuous. This may take, forexample, around 900 minutes (estimated), although the exact time dependsupon detection of the desired OD.

In some implementations, after growing the cells to the desired OD, aninducer is added to the first growth module 1502 for inducing the cells.In a particular example, 100 μl of inducer may be added, and the growthmodule 1502 may bring the temperature of the mixture up to 42° C. andhold for 15 minutes.

The cell stock 1512, after growth and induction, is transferred to thefirst filtration module 1504, in some implementations, for rendering thecells electrocompetent (1520 a) and to reduce the volume of the cellsfor transformation. In one example, a robotic handling system moves thevial of the cell stock 1512 from the rotating growth vial of the firstgrowth module 1502 to a vial holder of the first filtration module 1504.In another example, the robotic handling system pipettes cell stock 1512from the rotating growth vial of the first growth module 1502 anddelivers it to the first filtration module 1504. For example, thedisposable pipetting tip used to transfer the cell stock 1512 to thefirst growth module 1502 may be used to transfer the cell stock 1512from the first growth module 1502 to the first filtration module 1504.In some embodiments, prior to transferring the cell stock 1512 from thefirst growth module 1502 to the first filtration module 1504, the firstgrowth module 1502 is cooled to 4° C. so that the cell stock 1512 issimilarly reduced to this temperature. In a particular example, thetemperature of the first growth module 1502 may be reduced to about 4°C. over the span of about 8 minutes, and the growth module 1502 may holdthe temperature at 4° C. for about 15 minutes to ensure reduction intemperature of the cell stock 1512.

Prior to transferring the cell stock, in some implementations, a filterof the first filtration module 1504 is pre-washed using a wash solution.The wash solution, for example, may be supplied in a wash cartridge,such as the cartridge 106 described in relation to FIG. 1A.

The first filtration module 1504, for example, may be part of a dualfiltration module such as the filtration module 1250 described inrelation to FIGS. 12B and 12C. In a particular example, the firstfiltration module 1504 may be maintained at 4° C. during the washing andeluting process while transferring cell materials between an elutionvial and the first filtration module 1504.

In some implementations, upon rendering the cells electrocompetent atthe filtration module 1504, the cell stock 1512 is transferred to atransformation module 1506 (e.g., flow-through electroporation module)for transformation. In one example, a robotic handling system moves thevial of the cell stock 1512 from the vial holder of the first filtrationmodule 1504 to a reservoir of the flow-through electroporation module1506. In another example, the robotic handling system pipettes cellstock 1512 from the first filtration module 1502 or a temporaryreservoir and delivers it to the first filtration module 1504. In aparticular example, 400 μl of the concentrated cell stock 1512 from thefirst filtration module 1504 is transferred to a mixing reservoir priorto transfer to the transformation module 1506. For example, the cellstock 1512 may be transferred to a reservoir in a cartridge for mixingwith the assembled nucleic acids, then transferred by the robotichandling system using a pipette tip. In a particular example, thetransformation module is maintained at 4° C. The cell stock 1512 may betransformed, in an illustrative example, in about four minutes.

While the cells are growing and/or rendered electrocompetent, in someimplementations, a first oligo library 1514 a and the vector backbone1516 a are assembled using a nucleic acid assembly process to createassembled nucleic acids, e.g., using a thermal cycler and ligationprocess or in an nucleic acid assembly master mix (1522 a). Theassembled nucleic acids may be created at some point during the firstprocessing steps 1518 a, 1520 a of the first stage of the workflow 1500.Alternatively, assembled nucleic acids may be created in advance ofbeginning the first processing step 1518.

In some embodiments, the nucleic acids are assembled using a nucleicacid assembly module of the automated multi-module cell editinginstrument. For example, the robotic handling system may add the firstoligo library 1514 a and the vector backbone 1516 a from a libraryvessel in the reagent cartridge in the automated multi-module cellediting instrument to a nucleic acid assembly module (not illustrated),such as the nucleic acid assembly module 1710 g described in relation toFIG. 17B. In specific embodiments, the nucleic acid assembly performedin the nucleic acid assembly module is an isothermal nucleic acidassembly. The nucleic acid assembly mix, for example, may include in aparticular example 50 μl Gibson Assembly® Master Mix, 25 μl vectorbackbone 1516 a, and 25 μl oligo 1514 a. The nucleic acid assemblymodule may be held at room temperature or at another desiredtemperature.

In other embodiments, the nucleic acids are assembled externally to themulti-module cell editing instrument and added as a functioning sourcematerial. For example, a vial or tube of assembled nucleic acids may beadded to a reagent cartridge prior to activating the first step 1518 aof inoculation, growth and cell processing. In a particular example, 100μl of assembled nucleic acids are provided.

In other embodiments, the nucleic acids are introduced to the cells incomponents, and the machinery of the transformed cells will perform theassembly within the cell, e.g., using gap repair mechanisms in yeastcells.

In some implementations, the assembled nucleic acids are purified (1524a) prior to further use. Following assembly, for example, nucleic acids,may be transferred by the robotic handling system from the nucleic acidassembly module to a purification module (not shown). In otherembodiments, the nucleic acid assembly module may include purificationfeatures (e.g., a combination nucleic acid assembly and purificationmodule). In further embodiments, the assembled or separate nucleic acidsare purified externally to the multi-module cell editing instrument andadded as a functional source material. For example, a vial or tube ofpurified assembled nucleic acids may be added to a reagent cartridgewith the cell stock 1012 prior to activating the first step 1518 a ofcell processing.

In a particular example, 100 μl of assembled nucleic acids in nucleicacid assembly mix are assembled and subsequently purified. In someembodiments, magnetic beads are added to the nucleic acid assemblymodule, for example 180 μl of magnetic beads in a liquid suspension maybe added to the nucleic acid assembly module by the robotic handlingsystem. A magnet functionally coupled to the nucleic acid assemblymodule may be activated and the sample washed in 200 μl ethanol (e.g.,the robotic handling system may transfer ethanol to the nucleic acidassembly module). Liquid waste from this operation, in some embodiments,is transferred to a waste receptacle of the cartridge (e.g., by therobotic handling system using a same pipette tip as used in transferringthe ethanol). At this point, the de-salted assembled nucleic acids maybe transferred to a holding container, such as a reservoir of thecartridge. The desalted assembled nucleic acids may be held, for exampleat a temperature of 4° C. until cells are ready for transformation. In aparticular example, 100 μl of the assembled nucleic acids may be addedto the 400 μl of the concentrated cell stock 1512 in the mixingreservoir prior to transfer to the transformation module 1506. In someembodiments, the purification process may take about 16 minutes.

In some implementations, the assembled nucleic acids and cell stock 1512are added to the flow-through electroporation module 1506 and the cellstock 1512 is transformed (1526 a). The robotic handling system, forexample, may transfer the mixture of the cell stock 1512 and assemblednucleic acids to the flow-through electroporation module 1506 from amixing reservoir, e.g., using a pipette tip or through transferring avial or tube. In some embodiments, a built-in flow-throughelectroporation module such as the flow-through electroporation modulesdepicted in FIGS. 4A-4I, 5A-5H, 6, 8A-8U, and 9A-9C is used to transformthe cell stock 1512. In other embodiments, a cartridge-basedelectroporation module such as shown in FIGS. 10A-10C and 10E is used totransform the cell stock 1512. The electroporation module 1506, forexample, may be held at a temperature of 4° C. The electroporationprocess, in an illustrative example, may take about four minutes.

The transformed cell stock 1512 in some implementations is transferredto the second growth module 1508 for recovery (1528 a). In a particularexample, transformed cells undergo a recovery process in the secondgrowth module 1508 at a temperature of 30° C. The transformed cells, forexample, may be maintained in the second growth module 1508 for apredetermined period of time, e.g., about an hour for recovery.

In some implementations, a selective medium is transferred to the secondgrowth vial (not illustrated), and the cells are left to incubate for afurther period of time in a selection process. In an illustrativeexample, an antibiotic may be transferred to the second growth vial, andthe cells may incubate for an additional two hours at a temperature of30° C. to select for cells that have received the exogenous materials.

After recovery, the cells may be ready for either another round ofediting or for storage in a vessel, e.g., for further experimentsconducted outside of the automated cell processing environment.Alternatively, a portion of the cells may be transferred to a storageunit as cell library output, while another portion of the cells may beprepared for a second round of editing.

In some implementations, in preparation for a second round of editingthe transformed cells are transferred to the second filtration module1510 for media exchange and filtering (1530 a). Prior to transferringthe transformed cell stock, in some implementations, a filter of thesecond filtration module 1504 is pre-washed using a wash solution. Thewash solution, for example, may be supplied in a wash cartridge, such asthe cartridge 106 described in relation to FIG. 1A. The secondfiltration module 1510, for example, may be fluidly connected to thewash solution of the wash cartridge, as described in relation to FIG.12A.

The second filtration module 1510, for example, may be part of a dualfiltration module such as the filtration module 1250 described inrelation to FIGS. 12B and 12C. In a particular example, the secondfiltration module 1510 may be maintained at a predetermined temperature(e.g., 4° C.) during the washing and eluting process while transferringcell materials between an elution vial and the second filtration module1510. The output of this filtration process, in a particular example, isdeposited in a vial or tube to await further processing, e.g., transferto a transformation module. The vial or tube may be maintained in astorage unit at a temperature of 4° C.

The first stage of processing may take place during a single day. In oneillustrative embodiment, the first stage of processing is estimated totake under 19 hours to complete (e.g., about 18.7 hours). At this pointin the workflow 1500, in some implementations, new materials aremanually added to the automated multi-module cell editing instrument.For example, a new reagent cartridge may be added. Further, a new washcartridge, replacement filters, and/or replacement pipette tips may beadded to the automated multi-module cell editing instrument at thispoint. Further, in some embodiments, the filter module may undergo acleaning process and/or the solid and liquid waste units may be emptiedin preparation for the next round of processing In yet otherembodiments, the reagent cartridges may provide reagents for two or morecycles of editing, thus not requiring a change between two or moreediting rounds.

In some implementations, the second round of editing involves the samemodules 1502, 1504, 1506, 1508, and 1510, the same processing steps1518, 1520, 1522, 1524, 1526, 1528, and 1530, and the same temperatureand time ranges as the first processing stage described above. Forexample, the second oligo library 1514 b and the second vector backbone1516 b may be used to edit the transformed cells in much the same manneras described above. Although illustrated as a two-stage process, inother embodiments, up to two, four, six, eight, or more recursions maybe conducted to continue to edit the same cell stock 1512.

In other implementations, turning to FIG. 15B, a workflow 1540 involvesthe same modules 1502, 1504, 1506, 1508, and 1510 as well as the sameprocessing steps 1518, 1520, 1522, 1524, 1526, 1528, and 1530 for thefirst stage of process. However, unlike the workflow 1500 of FIG. 15A, asecond stage of the workflow 1540 of FIG. 15B involves a curing steps.“Curing” is a process in which a vector—for example the editing vectorused in the prior round of editing, the “engine” vector comprising theexpression sequence for the nuclease, or both—are eliminated from thetransformed cells. Curing can be accomplished by, e.g., cleaving theediting vector using a curing plasmid thereby rendering the editingand/or engine vector nonfunctional (exemplified in the workflow of FIG.15b ); diluting the vector in the cell population via cell growth (thatis, the more growth cycles the cells go through, the fewer daughtercells will retain the editing or engine vector(s)) (not shown), or by,e.g., utilizing a heat-sensitive origin of replication on the editing orengine vector (not shown). In one example, a “curing plasmid” may becontained within the reagent cartridge of the automated instrument, oradded manually to the instrument prior to the second stage ofprocessing. As with the workflow 1500, in some embodiments, the workflow1540 is performed based upon a script executed by a processing system ofthe automated multi-module cell editing instrument, such as theprocessing system 1810 of FIG. 18. The script, in a first example, maybe accessed via a machine-readable marker or tag added to the firstcartridge. In some embodiments, each processing stage is performed usinga separate script. For example, each cartridge may include an indicationof a script or a script itself for processing the contents of thecartridge. In this manner, for example, the second stage, involving thecuring cartridge, may be performed using a script designed for thesettings (e.g., temperatures, times, material quantities, etc.)appropriate for curing. The conditions for curing will depend on themechanism used for curing; that is, in this example, how the curingplasmid cleaves the editing and/or engine plasmid.

In some implementations, the second stage of the workflow 1540 begins byreceiving first-edited cells from the first stage of the workflow 1540at the first growth module 1502. For example, the first-edited cells mayhave been edited using a cell stock 1542, an oligo library 1544, and avector backbone 1546 through applying the steps 1518, 1520, 1522, 1524,1526, 1528, and 1530 as described in relation to the workflow 1500 ofFIG. 15A. The first-edited cell stock 1542, for example, may betransferred to the first growth module 1502 by a robotic handlingsystem. In one example, a robotic handling system adds a vial of thefirst-edited cell stock 1542 to a rotating growth vial of the firstgrowth module 1502. In another example, the robotic handling systempipettes first-edited cell stock 1542 from a receptacle of a storageunit and adds the cell stock 1542 to the rotating growth vial. The cellsmay have been maintained at a temperature of 4° C. at this point.

In some implementations, the first-edited cells are inoculated, grown,and monitored in the first growth module 1502 (1518 d). In a particularexample, an aliquot of the first-edited cell stock 1542 may betransferred to a rotating growth vial containing, e.g., 20 mL of growthmedium at a temperature of 30° C. to an OD of 0.50. The cell stock 1542added to the first growth module 1502 may be monitored over time until0.50 OD is sensed via the automated monitoring. Monitoring may beperiodic or continuous. This may take, for example, around 900 minutes(estimated), although the exact time depends upon detection of thedesired OD.

In some implementations, after growing to the desired OD, an inducer isadded to the first growth module 1502 for inducing the cells. In aparticular example, 100 μl of inducer may be added, and the growthmodule 1502 may bring the temperature of the mixture up to 42° C. andhold for 15 minutes.

The first-edited cell stock 1542, after growth and induction, istransferred to the first filtration module 1504, in someimplementations, for rendering the first-edited cells electrocompetent(1520 d). In one example, a robotic handling system moves the vial ofthe first-edited cell stock 1542 from the rotating growth vial of thefirst growth module 1502 to a vial holder of the first filtration module1504. In another example, the robotic handling system pipettesfirst-edited cell stock 1542 from the rotating growth vial of the firstgrowth module 1502 and delivers it to the first filtration module 1504.For example, the disposable pipetting tip used to transfer thefirst-edited cell stock 1542 to the first growth module 1502 may be usedto transfer the cell stock 1542 from the first growth module 1502 to thefirst filtration module 1504. In some embodiments, prior to transferringthe cell stock 1542 from the first growth module 1502 to the firstfiltration module 1504, the first growth module 1502 is cooled to 4° C.so that the cell stock 1542 is similarly reduced to this temperature. Ina particular example, the temperature of the first growth module 1502may be reduced to about 4° C. over the span of about 8 minutes, and thegrowth module 1502 may hold the temperature at 4° C. for about 15minutes to ensure reduction in temperature of the cell stock 1512.

Prior to transferring the first-edited cell stock 1542 to the filtrationmodule, in some implementations a filter of the first filtration module1504 is pre-washed using a wash solution. The wash solution, forexample, may be supplied in a wash cartridge, such as the cartridge 106described in relation to FIG. 1A. The first filtration module 1504, forexample, may be fluidly connected to the wash solution of the washcartridge.

The first filtration module 1504, for example, may be part of a dualfiltration module such as the filtration module 1250 described inrelation to FIGS. 12B and 12C. In a particular example, the firstfiltration module 1504 may be maintained at a predetermined temperature(e.g., 4° C.) during the washing and eluting process while transferringcell materials between an elution vial and the first filtration module1504.

In some implementations, upon rendering the first-edited cellselectrocompetent at the filtration module 1504 (1520 d), thefirst-edited cell stock 1542 is transferred to a transformation module1506 (e.g., FTEP module) for transformation. In one example, a robotichandling system moves the vial of the cell stock 1542 from the vialholder of the first filtration module 1504 to a reservoir of theflow-through electroporation module 1506. In another example, therobotic handling system pipettes cell stock 1542 from the firstfiltration module 1502 or a temporary reservoir and delivers it to thefirst filtration module 1504. In a particular example, 400 μl of theconcentrated cell stock 1542 from the first filtration module 1504 istransferred to a mixing reservoir prior to transfer to thetransformation module 1506. For example, the cell stock 1542 may betransferred to a reservoir in a cartridge for mixing with a curingplasmid 1550, then mixed and transferred by the robotic handling systemusing a pipette tip. In a particular example, the transformation module1506 is maintained at a predetermined temperature, e.g., 4° C. The cellstock 1542 may be transformed, in an illustrative example, in about fourminutes.

The transformed cell stock 1542, in some implementations, is transferredto the second growth module 1508 for recovery/curing (1528 d). In aparticular example 20 ml of transformed cells undergo a recovery processin the second growth module 1508 at a temperature of 30° C. Thetransformed cells, for example, may be maintained in the second growthmodule 1508 for about an hour for recovery. If another round of editingis desired, the first editing plasmid or vector is cured. If anotherround of editing is not desired, the first editing plasmid and theengine plasmid may be cured.

After recovery and curing, the cells may be ready for either anotherround of editing or for storage to be used in further research outsidethe automated cell processing instrument. For example, a portion of thecells may be transferred to a storage unit as cell library output, whileanother portion of the cells may be prepared for a second round ofediting.

In some implementations, in preparation for a second round of editing,the transformed cells are transferred to the second filtration module1510 for media exchange and filtering (1530 d) containing glycerol forrendering the cells electrocompetent. Prior to transferring thetransformed cell stock, in some implementations, a filter of the secondfiltration module 1504 is pre-washed using a wash solution. The washsolution, for example, may be supplied in a wash cartridge, such as thecartridge 106 described in relation to FIG. 1A. The second filtrationmodule 1510, for example, may be fluidly connected to the wash solutionof the wash cartridge, as described in relation to FIG. 12A.

The second filtration module 1510, for example, may be part of a dualfiltration module such as the filtration module 1250 described inrelation to FIGS. 12B and 12C. In a particular example, the secondfiltration module 1510 may be maintained at 4° C. during the washing andeluting process while transferring cell materials between an elutionvial and the second filtration module 1510. The output of thisfiltration process, in a particular example, are electrocompetent cellsdeposited in a vial or tube to await further processing. The vial ortube may be maintained in a storage unit at a temperature of 4° C.

Turning to FIG. 15C, a flow diagram illustrates a yeast workflow 1560involving two stages of processing having identical processing steps,resulting in two edits to a cell stock 1562. Each stage may operatebased upon a different cartridge of source materials. For example, afirst cartridge may include a first oligo library 1570 a and a firstvector backbone 1572 a. A second cartridge, introduced into theautomated multi-module cell editing instrument between processing stagesor prior to processing but in a different position than the firstcartridge, may include a second oligo library 1570 b and a second vectorbackbone 1572 b. Each cartridge may be considered as a “librarycartridge” for building a library of edited cells. Alternatively, a usermay add a container (e.g., vial or tube of the cell stock 1562 a to eachof the purchased cartridges included in a yeast cell kit.

The workflow 1560, in some embodiments, is performed based upon a scriptexecuted by a processing system of the automated multi-module cellediting instrument, such as the processing system 1810 of FIG. 18. Thescript, in a first example, may be accessed via a machine-readablemarker or tag added to the first cartridge. In some embodiments, eachprocessing stage is performed using a separate script. For example, eachcartridge may include an indication of a script or a script itself forprocessing the contents of the cartridge.

In some implementations, the first stage begins with introducing thecell stock 1562 into the first growth module 1502 for inoculation,growth, and monitoring (1518 e). In one example, a robotic handlingsystem adds a vial of the cell stock 1562 to a rotating growth vial ofthe first growth module 1502. In another example, the robotic handlingsystem pipettes cell stock 1562 from the first cartridge and adds thecell stock 1562 to the rotating growth vial. The cells may have beenmaintained at a temperature of 4° C. at this point. In a particularexample, 20 ml of cell stock may be grown within a rotating growth vialof the first growth module 1502 at a temperature of 30° C. to an OD of0.75. The cell stock 1512 added to the first growth module 1502 may beautomatically monitored over time within the growth module 1502 until0.75 OD is sensed via the automated monitoring. Monitoring may beperiodic or continuous.

In some implementations, an inducible expression system may be used.Thus, after growing to the desired OD, an inducer is added to the firstgrowth module 1502 for inducing the cells. The inducer could be a smallmolecule or a media exchange to a medium with a different sugar likegalactose.

The cell stock 1562, after growth and induction, is transferred to thefirst filtration module 1504, in some implementations, for exchangingmedia (1564 a). In one example, a robotic handling system moves the vialof the cell stock 1562 from the rotating growth vial of the first growthmodule 1502 to a vial holder of the first filtration module 1504. Inanother example, the robotic handling system pipettes cell stock 1562from the rotating growth vial of the first growth module 1502 anddelivers it to the first filtration module 1504. For example, thedisposable pipetting tip used to transfer the cell stock 1562 a to thefirst growth module 1502 may be used to transfer the cell stock 1562from the first growth module 1502 to the first filtration module 1504.In some embodiments, prior to transferring the cell stock 1562 from thefirst growth module 1502 to the first filtration module 1504, the firstgrowth module 1502 is cooled to 4° C. so that the cell stock 1562 issimilarly reduced to this temperature. In a particular example, thetemperature of the first growth module 1502 may be reduced to about 4°C. over the span of about 8 minutes, and the growth module 1502 may holdthe temperature at 4° C. for about 15 minutes to ensure reduction intemperature of the cell stock 1562. During media exchange, in anillustrative example, 0.4 ml of 1M sorbitol may be added to the cellstock 1562.

Prior to transferring the cell stock 1562, in some implementations, afilter of the first filtration module 1004 is pre-washed using a washsolution. The wash solution, for example, may be supplied in a washcartridge, such as the cartridge 106 described in relation to FIG. 1A.The first filtration module 1504, for example, may be fluidly connectedto the wash solution of the wash cartridge, as described in relation toFIG. 12A.

The first filtration module 1504, for example, may be part of a dualfiltration module such as the filtration module 1250 described inrelation to FIGS. 12B and 12C. In a particular example, the firstfiltration module 1504 may be maintained at 4° C. during the washing andeluting process while transferring cell materials between an elutionvial and the first filtration module 1504.

After the media exchange operation, in some implementations, the cellstock 1562 is transferred back to the first growth module 1502 forconditioning (1566 a). In one example, a robotic handling system movesthe vial of the cell stock 1562 from the first filtration module 1504 tothe first growth module 1502. In another example, the robotic handlingsystem pipettes cell stock 1562 from the first filtration module 1504and delivers it to the rotating growth vial of the first growth module1502. During conditioning, for example, 5 ml DTT/LIAc and 80 mM ofSorbitol may be added to the cell stock 1562. For example, the robotichandling system may transfer the DTT/LIAc and Sorbitol, individually orconcurrently, to the first growth module 1502. The cell stock 1562 maybe mixed with the DTT/LIAc and Sorbitol, for example, via the rotationof the rotating growth vial of the first growth module 1502. Duringconditioning, the cell stock 1562 may be maintained at a temperature of4° C.

In some implementations, after conditioning, the cell stock 1562 istransferred to the first filtration module 1504 for washing andpreparing the cells (1568). For example, the cells may be renderedelectrocompetent at this step. In one example, a robotic handling systemmoves the vial of the cell stock 1562 from the rotating growth vial ofthe first growth module 1502 to a vial holder of the first filtrationmodule 1504. In another example, the robotic handling system pipettescell stock 1562 from the rotating growth vial of the first growth module1502 and delivers it to the first filtration module 1004.

Prior to transferring the cell stock, in some implementations, a filterof the first filtration module 1504 is pre-washed using a wash solution.The wash solution, for example, may be supplied in a wash cartridge,such as the cartridge 106 described in relation to FIG. 1A. The firstfiltration module 1504, for example, may be fluidly connected to thewash solution of the wash cartridge, as described in relation to FIG.12A. In other embodiments, the same filter is used for renderingelectrocompetent as the filter used for media exchange at step 1564 a.In some embodiments, 1M sorbitol is used to render the yeast cellselectrocompetent.

In some implementations, upon rendering electrocompetent at thefiltration module 1504, the cell stock 1562 is transferred to atransformation module 1506 (e.g., flow-through electroporation module)for transformation. In one example, a robotic handling system moves thevial of the cell stock 1562 from the vial holder of the first filtrationmodule 1504 to a reservoir of the flow-through electroporation module1506. In another example, the robotic handling system pipettes cellstock 1562 from the filtration module 1504 or a temporary reservoir anddelivers it to the first filtration module 1504. In a particularexample, 400 μl of the concentrated cell stock 1562 from the firstfiltration module 1504 is transferred to a mixing reservoir prior totransfer to the transformation module 1506. For example, the cell stock1562 may be transferred to a reservoir in a cartridge for mixing withthe nucleic acid components (vector backbone and editingoligonucleotide), then mixed and transferred by the robotic handlingsystem using a pipette tip. Because the vector backbone and editingoligonucleotide are assembled in the cells (in vivo), a nucleic acidassembly module is not a necessary component for yeast editing. In aparticular example, the transformation module is maintained at 4° C.

In some implementations, the nucleic acids to be assembled and the cellstock 1562 is added to the FTEP module 1506 and the cell stock 1562 istransformed (1526 e). The robotic handling system, for example, maytransfer the mixture of the cell stock 1562 e and nucleic acid assemblyto the flow-through electroporation module 1506 from a mixing reservoir,e.g., using a pipette tip or through transferring a vial or tube. Insome embodiments, a built-in FTEP module such as the flow-throughelectroporation modules FIGS. 4A-4I, 5A-5G, 6, 8A-8U, 9A-9C, and 10A-10C(that is, single unit FTEPs) is used to transform the cell stock 1562 e.In other embodiments, a cartridge-based electroporation module is usedto transform the cell stock 1562 e. The FTEP module 1506, for example,may be held at a temperature of 4° C.

The transformed cell stock 1562 e, in some implementations, istransferred to the second growth module 1508 for recovery (1528 a). In aparticular example, 20 ml of transformed cells undergo a recoveryprocess in the second growth module 1508.

In some implementations, a selective medium, e.g. an auxotrophic growthmedium or a medium containing a drug, is transferred to the secondgrowth vial (not illustrated), and the cells are left to incubate for afurther period of time in a selection process. In an illustrativeexample, an antibiotic may be transferred to the second growth vial, andthe cells may incubate for an additional two hours at a temperature of30° C.

After recovery, the cells may be ready for either another round ofediting or for storage in a cell library. For example, a portion of thecells may be transferred to a storage unit as cell library output (1576a), while another portion of the cells may be prepared for a secondround of editing (1578 a). The cells may be stored, for example, at atemperature of 4° C.

In some implementations, in preparation for a second round of editing,the transformed cells are transferred to the second filtration module1510 for media exchange (1578 a). Prior to transferring the transformedcell stock 1562 a, in some implementations, a filter of the secondfiltration module 1504 is pre-washed using a wash solution. The washsolution, for example, may be supplied in a wash cartridge, such as thecartridge 106 described in relation to FIG. 1A. The second filtrationmodule 1510, for example, may be fluidly connected to the wash solutionof the wash cartridge, as described in relation to FIG. 12A.

The second filtration module 1510, for example, may be part of a dualfiltration module such as the filtration module 1250 described inrelation to FIGS. 12B and 12C. In a particular example, the secondfiltration module 1510 may be maintained at 4° C. during the washing andeluting process while transferring cell materials between an elutionvial and the second filtration module 1510.

In some implementations during the filtration process, an enzymaticpreparation is added to lyse the cell walls of the cell stock 1562 a.For example, a yeast lytic enzyme such as Zylomase® may be added to lysethe cell walls. The yeast lytic enzyme, in a particular example, may beincubated in the cell stock 1526 a for between 5-60 minutes at atemperature of 30° C. The output of this filtration process, in aparticular example, is deposited in a vial or tube to await furtherprocessing. The vial or tube may be maintained in a storage unit at atemperature of 4° C.

The first stage of processing may take place during a single day. Atthis point of the workflow 1560, in some implementations, new materialsare manually added to the automated multi-module cell editinginstrument. For example, new cell stock 1562 b and a new reagentcartridge may be added. Further, a new wash cartridge, replacementfilters, and/or replacement pipette tips may be added to the automatedmulti-module cell editing instrument at this point. Further, in someembodiments, the filter module may undergo a cleaning process and/or thesolid and liquid waste units may be emptied in preparation for the nextround of processing.

In some implementations, the second round of editing involves the samemodules 1502, 1504, 1506, 1508, and 1510, the same processing steps1518, 1564, 1566, 1526, 1528, and 1576 and/or 1578, and the sameconditions (e.g., temperatures, time ranges, etc.) as the firstprocessing stage described above. For example, the second oligo library1570 b and the second vector backbone 1572 b may be used to edit acombination of the transformed cells in much the same manner asdescribed above. Although illustrated as a two-stage process, in otherembodiments, up to two, three, four, six, eight, or more recursions maybe conducted to continue to edit the cell stock 1562.

Alternative Embodiments of Instrument Architecture

FIGS. 17A and 17B illustrate exemplary alternative embodiments ofautomated multi-module cell editing instruments for performing automatedcell processing, e.g., editing in multiple cells in a single cycle. Theautomated multi-module cell editing instruments, for example, may bedesktop instruments designed for use within a laboratory environment.The automated multi-module cell editing instruments may incorporate bothreusable and disposable elements for performing various stagedoperations in conducting automated genome cleavage and/or editing incells.

FIG. 17A is a block diagram of a first example instrument 1700 forperforming automated cell processing, e.g., editing in multiple cells ina single cycle according to one embodiment of the disclosure. In someimplementations, the instrument 1700 includes a deck, a reagent supplyreceptacle 1704 for introducing DNA sample components to the instrument1700, a cell supply receptacle 1706 for introducing cells to theinstrument 1700, and a robot handling system 1708 for moving materialsbetween modules (for example, modules 1710 a, 1710 b, 1710 c, 1710 d)receptacles (for example, receptacles 1704, 1706, 1712 a-c, 1722, 1724,and 1726), and storage units (e.g., units 1718, 1728, and 1714) of theinstrument 1700 to perform the automated cell processing. Uponcompletion of editing of the cell supply 1706, in some embodiments, celloutput 1712 may be transferred by the robot handling system 1708 to astorage unit 1714 for temporary storage and later retrieval.

The robotic handling system 1708, for example, may include an airdisplacement pump to transfer liquids from the various material sourcesto the various modules 1710 a-d and storage unit 1714. In otherembodiments, the robotic handling system 1708 may include a pick andplace head to transfer containers of source materials (e.g., tubes) froma supply cartridge (not illustrated, discussed in relation to FIG. 1A)to the various modules 1710. In some embodiments, one or more cameras orother optical sensors (not shown), confirm proper gantry movement andposition.

In some embodiments, the robotic handling system 1708 uses disposabletransfer tips provided in a transfer tip supply 1716 to transfer sourcematerials, reagents 1704 (e.g., for nucleic acid assembly), and cells1706 within the instrument 1700. Used transfer tips, for example, may bediscarded in a solid waste unit 1718. In some implementations, the solidwaste unit 1718 contains a kicker to remove tubes from the pick andplace head of robotic handling system 1708.

In some embodiments, the instrument 1700 includes electroporatorcuvettes with sippers that connect to an air displacement pump. In someimplementations, cells 1706 and reagent 1704 are aspirated into theelectroporation cuvette through a sipper, and the cuvette is moved toone or more modules 1710 a-d of the instrument 1700.

In some implementations, the instrument 1700 is controlled by aprocessing system 1720 such as the processing system 1810 of FIG. 18.The processing system 1720 may be configured to operate the instrument1700 based on user input. The processing system 1720 may control thetiming, duration, temperature and other operations of the variousmodules 1710 of the instrument 1700. The processing system 1720 may beconnected to a power source (not shown) for the operation of theinstrument 1700.

In some embodiments, instrument 1700 includes an FTEP device 1710 c tointroduce nucleic acid(s) into the cells 1706. For example, the robotichandling system 1708 may transfer the reagent 1704 and cells 1706 to theFTEP device 1710 c. The FTEP device 1710 conducts cell transformation ortransfection via electroporation. The processing system 1720 may controltemperature and operation of the FTEP device 1710 c. In someimplementations, the processing system 1720 effects operation of theFTEP device 1710 c according to one or more variable controls set by auser.

Following transformation, in some implementations, the cells may betransferred to a recovery module 1710 d. In some embodiments, therecovery module 1710 d is a combination recovery and induction ofediting module. In the recovery module 1710 d, the cells are allowed torecover, express the nucleic acids and, in an inducible nuclease system,a nuclease or guide RNA is induced in the cells, e.g., by means oftemporally-controlled induction such as, in some examples, chemical,light, viral, or temperature induction or the introduction of an inducermolecule 1724 for expression of the nuclease.

Following editing, in some implementations the cells are transferred tothe storage unit 1714, where the cells can be stored as cell output 1712a-d until the cells are removed for further study or retrieval of anedited cell population, e.g., an edited cell library.

In some implementations the instrument 1700 is designed for recursivegenome editing, where multiple edits are sequentially introduced intogenomes inside the cells of a cell population. In some implementations,the reagent supply 1704 is replenished prior to accessing cell output1712 a-d from the storage unit for recursive processing. In otherimplementations, multiple reagent supplies 1704 and/or large volumesthereof may be introduced into the instrument 1700 such that userinteraction is not necessarily required prior to a subsequent processingcycle.

A portion of a cell output 1712 a, in some embodiments, is transferredto an automated cell growth module 1710 a. For example, all of the celloutput 1712 a may be transferred, or only an aliquot may be transferredsuch that the instrument retains incrementally modified samples. Thecell growth module 1710 a, in some implementations, measures the OD ofthe cells during growth to ensure they are at a desired concentrationprior to induction of editing. Other measures of cell density andphysiological state that can be used include but are not limited to, pH,dissolved oxygen, released enzymes, acoustic properties, and electricalproperties.

To reduce the background of cells that have not received a genome edit,in some embodiments the growth module 1710 a performs a selectionprocess to enrich for the edited cells using a selective growth medium1726. For example, the introduced nucleic acid can include a gene thatconfers antibiotic resistance or some other selectable marker. In someimplementations, multiple selective genes or markers 1726 may beintroduced into the cells during recursive editing. For example,alternating the introduction of selectable markers for sequential roundsof editing can eliminate the background of unedited cells and allowmultiple cycles of the instrument 1700 to select for cells havingsequential genome edits. Suitable antibiotic resistance genes include,but are not limited to, genes such as ampicillin-resistance gene,tetracycline-resistance gene, kanamycin-resistance gene,neomycin-resistance gene, canavanine-resistance gene,blasticidin-resistance gene, hygromycin-resistance gene,puromycin-resistance gene, and chloramphenicol-resistance gene.

From the growth module 1710 a, the cells may be transferred to afiltration module 1710 b. The filtration module 1710 b or,alternatively, a cell wash and concentration module, may enable mediaexchange. In some embodiments, removing dead cell background is aidedusing lytic enhancers such as detergents, osmotic stress, temperature,enzymes, proteases, bacteriophage, reducing agents, or chaotropes. Inother embodiments, cell removal and/or media exchange is used to reducedead cell background. Waste product from the filtration module 1710 b,in some embodiments, is collected in a liquid waste unit 1728.

After filtration, the cells may be presented to the FTEP device(transformation module) 1710 c, and then to the recovery module 1710 dand finally to the storage unit 1714 as detailed above.

Turning to FIG. 17B, similar to FIG. 17A, a second exemplary instrument1740 for performing automated genome cleavage and/or editing in multiplecells in a single cycle includes the deck 1702, the reagent supplyreceptacle 1704 for introducing one or more nucleic acid components tothe instrument 1740, the cell supply receptacle 1706 for introducingcells to the instrument 1740, and the robot handling system 1708 formoving materials between modules (for example, modules 1710 a, 1710 b,1710 c, 1710 f, 1710 g, 1710 m, and 1710 h), receptacles (for example,receptacles 1704 1706, 1712 a-c, 1724, 1742, 1744, and 1746), andstorage units (e.g., units 1714, 1718, and 1728) of the instrument 1740to perform the automated cell processing. Upon completion of processingof the cell supply 1706, in some embodiments, cell output 1712 a-c maybe transferred by the robot handling system 1708 to the storage unit1714 for temporary storage and later retrieval.

In some embodiments, the robotic handling system 1708 uses disposabletransfer tips provided in the transfer tip supply 1716 to transfersource materials, a vector backbone 1742, editing oligos 1744, reagents1704 (e.g., for nucleic acid assembly, nucleic acid purification, torender cells electrocompetent, etc.), and cells 1706 within theinstrument 1740, as described in relation to FIG. 17A.

As described in relation to FIG. 17A, in some implementations, theinstrument 1740 is controlled by the processing system 1720 such as theprocessing system 1810 of FIG. 18.

The instrument 1740, in some embodiments, includes a nucleic acidassembly module 1710 g, and in certain exemplary automated multi-modulecell editing instruments, the nucleic acid assembly module 1710 g mayperform in some embodiments nucleic acid assembly.

In some embodiments, after assembly of the nucleic acids, the nucleicacids (e.g., in the example of a nucleic acid assembly, the nucleic acidassembly mix (nucleic acids+nucleic acid assembly reagents)) aretransferred to a purification module 1710 h. Here, unwanted componentsof the nucleic acid assembly mixture are removed (e.g., salts) and, incertain embodiments, the assembled nucleic acids are concentrated. Forexample, in an illustrative embodiment, in the purification module 1710h, the nucleic acid assembly mix may be combined with a no-salt bufferand magnetic beads, such as Solid Phase Reversible Immobilization (SPRI)magnetic beads or AMPure™ beads. The nucleic acid assembly mix may beincubated for sufficient time (e.g., 30 seconds to 10 minutes) for theassembled nucleic acids to bind to the magnetic beads. In someembodiments, the purification module includes a magnet configured toengage the magnetic beads. The magnet may be engaged so that thesupernatant may be removed from the bound assembled nucleic acids and sothat the bound assembled nucleic acids can be washed with, e.g., 80%ethanol. Again, the magnet may be engaged and the 80% ethanol washsolution removed. The magnetic bead/assembled nucleic acids may beallowed to dry, then the assembled nucleic acids may be eluted and themagnet may again be engaged, this time to sequester the beads and toremove the supernatant that contains the eluted assembled nucleic acids.The assembled nucleic acids may then be transferred to thetransformation module (e.g., electroporator in a preferred embodiment).The transformation module may already contain the electrocompetent cellsupon transfer.

Instrument 1740 includes an FTEP device 1710 c for introduction of thenucleic acid(s) into the cells 1706, as described in relation to FIG.17A. However, in this circumstance, the assembled nucleic acids 1704,output from the purification module 1710 h, are transferred to the FTEPdevice 1710 c to combine with the cells 1706.

Following transformation in the FTEP device 1710 c, in someimplementations, the cells may be transferred to a recovery module 1710m. In the recovery module 1710 e, the cells are allowed to recover,express the exogenous nucleic acids, electroporated into the cells and,in an inducible system, the nuclease or other editing component such asthe guide nucleic acid is induced, e.g., by means oftemporally-controlled induction such as, in some examples, chemical,light, viral, or temperature induction or the introduction of theinducer molecule for expression of the editing component.

Following recovery, in some implementations the cells are transferred toan editing module 1710 f. The editing module 1710 f provides appropriateconditions to induce editing of the cells' genomes, e.g., throughexpression of the introduced nucleic acids and the induction of aninducible nuclease or guide nucleic acid. The editing components may be,in some examples, chemically induced, biologically induced (e.g., viainducible promoter) virally induced, light induced, temperature induced,and/or heat induced within the editing module 1710 f.

Following editing, in some implementations, the cells are transferred tothe storage unit 1714 as described in relation to FIG. 17A.

In some implementations, the instrument 1740 is designed for recursivegenome editing, where multiple edits are sequentially introduced intogenomes of cells in a cell population. In some implementations, thereagent supply 1704 is replenished prior to accessing cell output 1712from the storage unit for recursive processing. For example, additionalvector backbone 1742 and/or editing oligos 1744 may be introduced intothe instrument 1740 for assembly and preparation via the nucleic acidassembly module 1710 g and the purification module 1710 h. In otherimplementations, multiple vector backbone volumes 1742 and/or editingoligos 1744 may be introduced into the instrument 1700 such that userinteraction is not necessarily required prior to a subsequent processingcycle. For each subsequent cycle, the vector backbone 1742 and/orediting oligos 1744 may change. Upon preparation of the nucleic acidassembly, the nucleic acid assembly may be provided in the reagentsupply 1704 or another storage region.

A portion of a cell output 1712 a, in some embodiments, is transferredto the automated cell growth module 1710 a, as discussed in relation toFIG. 17A.

To reduce background of cells that have not received a genome edit, insome embodiments, the growth module 1710 a performs a selection processto enrich for the edited cells using a selective growth medium 1726, asdiscussed in relation to FIG. 17A.

From the growth module 1710 a, the cells may be transferred to thefiltration module 1710 b, as discussed in relation to FIG. 17A. Asillustrated, eluant from an eluting supply 1746 (e.g. buffer, glycerol)may be transferred into the filtration module 1710 b for media exchange.

After filtration, the cells may be transferred to the FTEP device 1710 cfor transformation, and then to the recovery module 1710 m, and theediting module 1710 f and finally to the storage unit 1714 as detailedabove.

In some embodiments, the automated multi-module cell editing instrumentsof FIGS. 17A and/or 17B contain one or more replaceable supplycartridges and a robotic handling system, as discussed in relation toFIGS. 1A and 1B. Each cartridge may contain one or more of a nucleicacid assembly mix, oligonucleotides, vector, growth media, selectionagent (e.g., antibiotics), inducing agent, nucleic acid purificationreagents such as Solid Phase Reversible Immobilization (SPRI) beads,ethanol, and 10% glycerol.

Although the exemplary instruments 1700, 1740 are illustrated asincluding a particular arrangement of modules 1710, these arrangementsare for illustrative purposes only. For example, in other embodiments,more or fewer modules 1710 may be included within each of theinstruments 1700, 1740. Also, different modules may be included in theinstrument, such as, e.g., a module that facilitates cell fusion forproviding, e.g., hybridomas, a module that amplifies nucleic acidsbefore assembly, and/or a module that facilitates protein expressionand/or secretion. Further, certain modules 1710 may be replicated withincertain embodiments, such as the duplicate cell growth modules 110 a,110 b of FIG. 1A. Each of the instruments 1700 and 1740, in anotherexample, may be designed to accept a media cartridge such as thecartridges 104 and 106 of FIG. 1A. Further modifications are possible.

Control System for an Automated Multi-module cell Editing Instrument

Turning to FIG. 16, a screen shot illustrates an example graphical userinterface (GUI) 1600 for interfacing with an automated multi-module cellediting instrument. The interface, for example, may be presented on thedisplay 236 of FIGS. 2C and 2D. In one example, the GUI 1600 may bepresented by the processing system 1810 of FIG. 18 on the touch screen1816.

In some implementations, the GUI 1600 is divided into a number ofinformation and data entry panes, such as a protocol pane 1602, atemperature pane 1606, an electroporation pane 1608, and a cell growthpane 1610. Further panes are possible. For example, in some embodimentsthe GUI 1600 includes a pane for each module, such as, in some examples,one or more of each of a nucleic acid assembly module, a purificationmodule, a cell growth module, a filtration module, a transformationmodule, an editing module, and a recovery module. The lower panes of theGUI 1600, in some embodiments, represent modules applicable to thepresent work flow (e.g., as selected in the protocol pane 1602 or asdesignated within a script loaded through a script interface (notillustrated)). In some embodiments, a scroll or paging feature may allowthe user to access additional panes not illustrated within the screenshot of FIG. 16.

The GUI 1600, in some embodiments, includes a series of controls 1620for accessing various screens such as the illustrated screen shot (e.g.,through using a home control 1620 a). For example, through selecting anediting control 1620 b, the user may be provided the option to provideone, two or a series of cell editing steps. Through selecting a scriptcontrol 1620 c, the user may be provided the opportunity to add a newediting script or alter an existing editing script. The user in someembodiments, may select a help control 1620 d to obtain furtherinformation regarding the features of the GUI 1600 and the automatedmulti-module cell editing instrument. In some implementations, the userselects a settings control 1620 e to access settings options for desiredprocesses and/or the GUI 1600 such as, in some examples, time zone,language, units, network access options. A power control 1620 f, whenselected, allows the user to power down the automated multi-module cellediting instrument.

Turning to the protocol pane 1602, in some implementations, a userselects a protocol (e.g., script or work flow) for execution by theautomated multi-module cell editing instrument by entering the protocolin a protocol entry field 1612 (or, alternatively, drop-down menu). Inother embodiments, the protocol may be selected through a separate userinterface screen, accessed for example by selecting the script control1620 b. In another example, the automated multi-module cell editinginstrument may select the protocol and present it in the protocol entryfield 1612. For example, the processing system of the automatedmulti-module cell editing instrument may scan machine-readable indiciapositioned on one or more cartridges loaded into the automatedmulti-module cell editing instrument to determine the appropriateprotocol. As illustrated, the “Microbe Kit 1 (1.0.2)” protocol has beenselected, which may correspond to a kit of cartridges and otherdisposable supplies purchased for use with the automated multi-modulecell editing instrument.

In some implementations, the protocol pane 1602 further includes a startcontrol 1614 a and a stop control 1614 b to control execution of theprotocol presented in the protocol entry field 1612. The GUI 1600 may beprovided on a touch screen interface, for example, where touch selectionof the start control 1614 a starts cell processing, and selection of thestop control 1614 b stops cell processing.

Turning to the run status pane 1604, in some implementations a chart1616 illustrates stages of the processing of the protocol identified inthe protocol pane 1602. For example, a portion of run completion 1618 ais illustrated in blue, while a portion of current stage 1618 b isillustrated in green, and any errors 1618 c are flagged with markersextending from the point in time along the course of the portion of therun completion 1618 a where the error occurred. A message region 1618 dpresents a percentage of run completed, a percentage of stage completed,and a total number of errors. In some embodiments, upon selection of thechart 1616, the user may be presented with greater details regarding therun status such as, in some examples, identification of the type oferror, a name of the current processing stage (e.g., nucleic acidassembly, purification, cell growth, filtration, transformation,recovery, editing, etc.), and a listing of processing stages within therun. Further, in some embodiments a run completion time messageindicates a date and time at which the run is estimated to complete. Therun, in some examples, may be indicative of a single cell editingprocess or a series of recursive cell editing processes scheduled forexecution without user intervention. In some embodiments (not shown),the run status pane 1604 additionally illustrates an estimated time atwhich user intervention will be required (e.g., cartridge replacement,solid waste disposal, liquid waste disposal, etc.).

In some implementations, the run status pane 1604 includes a pausecontrol 1624 for pausing cell processing. The user may select to pausethe current run, for example, to correct for an identified error or toconduct manual intervention such as waste removal.

The temperature pane 1606, in some embodiments, illustrates a series oficons 1126 with corresponding messages 1628 indicating temperaturesettings for various apparatus of the automated multi-module cellediting instrument. The icons, from left to right, may represent an FTEPmodule 1626 a (e.g., FTEP device associated with the reagent cartridge110 c of FIG. 1A), a purification module 1626 b, a first growth module1626 c, a second growth module 1626 d, and a filtration module 1626 e.The corresponding messages 1628 a-e identify a present temperature, lowtemperature, and high temperature of the corresponding module (e.g., forthis stage or this run). In selecting one of the icons 1626, in someembodiments, a graphic display of temperature of time may be reviewed.

Beneath the temperature pane, in some implementations, a series of panesidentify present status of a number of modules. For example, theelectroporation pane 1608 represents status of a transformation module,while the cell growth pane 1610 represents the status of a growthmodule. In some embodiments, the panes presented here identify status ofa presently operational module (e.g., the module involved in cellprocessing in the current stage) as well as the status of any moduleswhich have already been utilized during the current run (as illustrated,for example, in the run status pane 1604). Past status information, forexample, may present to the user information regarding the parametersused in the prior stage(s) of cell processing.

Turning to the electroporation pane 1608, in some implementations,operational parameters 1630 a of volts, milliamps, and joules arepresented. Additionally, a status message 1632 a may identify additionalinformation regarding the functioning of the transformation module suchas, in some examples, an error status, a time remaining for processing,or contents of the module (e.g., materials added to the module). In someimplementations, an icon 1634 a above the status message 1632 a will bepresented in an active mode (e.g., colorful, “lit up”, in bold, etc.)when the corresponding module is actively processing. Selection of theicon 1634 a, in some embodiments, causes presentation of a graphicdisplay of detailed information regarding the operational parameters1630 a.

Turning to the cell growth pane 1610, in some implementations,operational parameters 1630 b of OD and hours of growth are presented.Additionally, a status message 1632 b may identify additionalinformation regarding the functioning of the growth module such as, insome examples, an error status, a time remaining for processing, orcontents of the module (e.g., materials added to the module). In someimplementations, an icon 1634 b above the status message 1632 b will bepresented in an active mode (e.g., colorful, “lit up”, in bold, etc.)when the corresponding module is actively processing. Selection of theicon 1634 b, in some embodiments, causes presentation of a graphicdisplay of detailed information regarding the operational parameters1630 b.

Next, a hardware description of an example processing system andprocessing environment according to exemplary embodiments is describedwith reference to FIG. 18. In FIG. 18, the processing system 1810includes a CPU 1808 which performs a portion of the processes describedabove. For example, the CPU 1808 may manage the processing stages of themethod 1400 of FIG. 14 and/or the workflows of FIGS. 15A-C. The processdata and, scripts, instructions, and/or user settings may be stored inmemory 1802. These process data and, scripts, instructions, and/or usersettings may also be stored on a storage medium disk 1804 such as aportable storage medium (e.g., USB drive, optical disk drive, etc.) ormay be stored remotely. For example, the process data and, scripts,instructions, and/or user settings may be stored in a locationaccessible to the processing system 1810 via a network 1828. Further,the claimed advancements are not limited by the form of thecomputer-readable media on which the instructions of the inventiveprocess are stored. For example, the instructions may be stored in FLASHmemory, RAM, ROM, or any other information processing device with whichthe processing system 1810 communicates, such as a server, computer,smart phone, or other hand-held computing device.

Further, components of the claimed advancements may be provided as autility application, background daemon, or component of an operatingsystem, or combination thereof, executing in conjunction with CPU 1808and an operating system such as with other computing systems known tothose skilled in the art.

CPU 1808 may be an ARM processor, system-on-a-chip (SOC),microprocessor, microcontroller, digital signal processor (DSP), or maybe other processor types that would be recognized by one of ordinaryskill in the art. Further, CPU 1808 may be implemented as multipleprocessors cooperatively working in parallel to perform the instructionsof the inventive processes described above.

The processing system 1810 is part of a processing environment 1800. Theprocessing system 1810 in FIG. 18 also includes a network controller1806 for interfacing with the network 1828 to access additional elementswithin the processing environment 1800. As can be appreciated, thenetwork 1828 can be a public network, such as the Internet, or a privatenetwork such as an LAN or WAN network, or any combination thereof andcan also include PSTN or ISDN sub-networks. The network 1828 can bewireless such as a cellular network including EDGE, 3G and 4G wirelesscellular systems. The wireless network can also be Wi-Fi, Bluetooth, orany other wireless form of communication that is known.

The processing system 1810 further includes a general purpose I/Ointerface 1812 interfacing with a user interface (e.g., touch screen)1816, one or more sensors 1814, and one or more peripheral devices 1818.The peripheral I/O devices 1818 may include, in some examples, a videorecording system, an audio recording system, microphone, externalstorage devices, and/or external speaker systems. The one or moresensors 1814 may include one or more of a gyroscope, an accelerometer, agravity sensor, a linear accelerometer, a global positioning system, abar code scanner, a QR code scanner, an RFID scanner, a temperaturemonitor, and a lighting system or lighting element.

The general purpose storage controller 1824 connects the storage mediumdisk 1804 with communication bus 1840, such as a parallel bus or aserial bus such as a Universal Serial Bus (USB), or similar, forinterconnecting all of the components of the processing system. Adescription of the general features and functionality of the storagecontroller 1824, network controller 1806, and general purpose I/Ointerface 1812 is omitted herein for brevity as these features areknown.

The processing system 1810, in some embodiments, includes one or moreonboard and/or peripheral sensors 1814. The sensors 1814, for example,can be incorporated directly into the internal electronics and/or ahousing of the automated multi-module processing instrument. A portionof the sensors 1814 can be in direct physical contact with the I/Ointerface 1812, e.g., via a wire; or in wireless contact e.g., via aBluetooth, Wi-Fi or NFC connection. For example, a wirelesscommunications controller 1826 may enable communications between one ormore wireless sensors 1814 and the I/O interface 1812. Furthermore, oneor more sensors 1814 may be in indirect contact e.g., via intermediaryservers or storage devices that are based in the network 1828; or in(wired, wireless or indirect) contact with a signal accumulatorsomewhere within the automated multi-module cell editing instrument,which in turn is in (wired or wireless or indirect) contact with the I/Ointerface 1812.

A group of sensors 1814 communicating with the I/O interface 1812 may beused in combination to gather a given signal type from multiple placesin order to generate a more complete map of signals. One or more sensors1814 communicating with the I/O interface 1812 can be used as acomparator or verification element, for example to filter, cancel, orreject other signals.

In some embodiments, the processing environment 1800 includes acomputing device 1838 communicating with the processing system 1810 viathe wireless communications controller 1826. For example, the wirelesscommunications controller 1826 may enable the exchange of emailmessages, text messages, and/or software application alerts designatedto a smart phone or other personal computing device of a user.

The processing environment 1800, in some implementations, includes arobotic material handling system 1822. The processing system 1810 mayinclude a robotics controller 1820 for issuing control signals toactuate elements of the robotic material handling system, such asmanipulating a position of a gantry, lowering or raising a sipper orpipettor element, and/or actuating pumps and valves to cause liquidtransfer between a sipper/pipettor and various vessels (e.g., chambers,vials, etc.) in the automated multi-module cell editing instrument. Therobotics controller 1820, in some examples, may include a hardwaredriver, firmware element, and/or one or more algorithms or softwarepackages for interfacing the processing system 1810 with the roboticsmaterial handling system 1822.

In some implementations, the processing environment 1810 includes one ormore module interfaces 1832, such as, in some examples, one or moresensor interfaces, power control interfaces, valve and pump interfaces,and/or actuator interfaces for activating and controlling processing ofeach module of the automated multi-module processing system. Forexample, the module interfaces 1832 may include an actuator interfacefor the drive motor of rotating cell growth device 1350 (FIGS. 8C and8D) and a sensor interface for the detector board 1372 that sensesoptical density of cell growth within rotating growth vial 1300. Amodule controller 1830, in some embodiments, is configured to interfacewith the module interfaces 1832. The module controller 1830 may includeone or many controllers (e.g., possibly one controller per module,although some modules may share a single controller). The modulecontroller 1830, in some examples, may include a hardware driver,firmware element, and/or one or more algorithms or software packages forinterfacing the processing system 1810 with the module interfaces 1832.

The processing environment 1810, in some implementations, includes athermal management system 1836 for controlling climate conditions withinthe housing of the automated multi-module processing system. The thermalmanagement system 1836 may additional control climate conditions withinone or more modules of the automated multi-module cell editinginstrument. The processing system 1810, in some embodiments, includes atemperature controller 1834 for interfacing with the thermal managementsystem 1836. The temperature controller 1834, in some examples, mayinclude a hardware driver, firmware element, and/or one or morealgorithms or software packages for interfacing the processing system1810 with the thermal management system 1836.

Production of Cell Libraries Using Automated Editing Methods, Modules,Instruments and Systems

In one aspect, the present disclosure provides automated editingmethods, modules, instruments, and automated multi-module cell editinginstruments for creating a library of cells that vary the expression,levels and/or activity of RNAs and/or proteins of interest in variouscell types using various editing strategies, as described herein in moredetail. Accordingly, the disclosure is intended to cover edited celllibraries created by the automated editing methods, automatedmulti-module cell editing instruments of the disclosure. These celllibraries may have different targeted edits, including but not limitedto gene knockouts, gene knock-ins, insertions, deletions, singlenucleotide edits, short tandem repeat edits, frameshifts, triplet codonexpansion, and the like in cells of various organisms. These edits canbe directed to coding or non-coding regions of the genome, and arepreferably rationally designed.

In other aspects, the present disclosure provides automated editingmethods, automated multi-module cell editing instruments for creating alibrary of cells that vary DNA-linked processes. For example, the celllibrary may include individual cells having edits in DNA binding sitesto interfere with DNA binding of regulatory elements that modulateexpression of selected genes. In addition, cell libraries may includeedits in genomic DNA that impact on cellular processes such asheterochromatin formation, switch-class recombination and VDJrecombination.

In specific aspects, the cell libraries are created using multiplexedediting of individual cells within a cell population, with multiplecells within a cell population are edited in a single round of editing,i.e., multiple changes within the cells of the cell library are in asingle automated operation. The libraries that can be created in asingle multiplexed automated operation can comprise as many as 500edited cells, 1000 edited cells, 2000 edited cells, 5000 edited cells,10,000 edited cells, 50,000 edited cells, 100,000 edited cells, 200,000edited cells, 300,000 edited cells, 400,000 edited cells, 500,000 editedcells, 600,000 edited cells, 700,000 edited cells, 800,000 edited cells,900,000 edited cells, 1,000,000 edited cells, 2,000,000 edited cells,3,000,000 edited cells, 4,000,000 edited cells, 5,000,000 edited cells,6,000,000 edited cells, 7,000,000 edited cells, 8,000,000 edited cells,9,000,000 edited cells, 10,000,000 edited cells or more.

In other specific aspects, the cell libraries are created usingrecursive editing of individual cells within a cell population, withedits being added to the individual cells in two or more rounds ofediting. The use of recursive editing results in the amalgamation of twoor more edits targeting two or more sites in the genome in individualcells of the library. The libraries that can be created in an automatedrecursive operation can comprise as many as 500 edited cells, 1000edited cells, 2000 edited cells, 5000 edited cells, 10,000 edited cells,50,000 edited cells, 100,000 edited cells, 200,000 edited cells, 300,000edited cells, 400,000 edited cells, 500,000 edited cells, 600,000 editedcells, 700,000 edited cells, 800,000 edited cells, 900,000 edited cells,1,000,000 edited cells, 2,000,000 edited cells, 3,000,000 edited cells,4,000,000 edited cells, 5,000,000 edited cells, 6,000,000 edited cells,7,000,000 edited cells, 8,000,000 edited cells, 9,000,000 edited cells,10,000,000 edited cells or more.

Examples of non-automated editing strategies that can be modified basedon the present specification to utilize the automated systems can befound, e.g., U.S. Pat. Nos. 8,110,360, 8,332,160, 9,988,624,20170316353, and 20120277120.

In specific aspects, recursive editing can be used to first create acell phenotype, and then later rounds of editing used to reverse thephenotype and/or accelerate other cell properties.

In some aspects, the cell library comprises edits for the creation ofunnatural amino acids in a cell.

In specific aspects, the disclosure provides edited cell librarieshaving edits in one or more regulatory elements created using theautomated editing methods, automated multi-module cell editinginstruments of the disclosure. The term “regulatory element” refers tonucleic acid molecules that can influence the transcription and/ortranslation of an operably linked coding sequence in a particularenvironment and/or context. This term is intended to include allelements that promote or regulate transcription, and RNA stabilityincluding promoters, core elements required for basic interaction of RNApolymerase and transcription factors, upstream elements, enhancers, andresponse elements (see, e.g., Lewin, “Genes V” (Oxford University Press,Oxford) pages 847-873). Exemplary regulatory elements in prokaryotesinclude, but are not limited to, promoters, operator sequences and aribosome binding sites. Regulatory elements that are used in eukaryoticcells may include, but are not limited to, promoters, enhancers,insulators, splicing signals and polyadenylation signals.

Preferably, the edited cell library includes rationally designed editsthat are designed based on predictions of protein structure, expressionand/or activity in a particular cell type. For example, rational designmay be based on a system-wide biophysical model of genome editing with aparticular nuclease and gene regulation to predict how different editingparameters including nuclease expression and/or binding, growthconditions, and other experimental conditions collectively control thedynamics of nuclease editing. See, e.g., Farasat and Salis, PLoS ComputBiol., 29:12(1):e1004724 (2016).

In one aspect, the present disclosure provides the creation of a libraryof edited cells with various rationally designed regulatory sequencescreated using the automated editing instrumentation, systems and methodsof the invention. For example, the edited cell library can includeprokaryotic cell populations created using set of constitutive and/orinducible promoters, enhancer sequences, operator sequences and/orribosome binding sites. In another example, the edited cell library caninclude eukaryotic sequences created using a set of constitutive and/orinducible promoters, enhancer sequences, operator sequences, and/ordifferent Kozak sequences for expression of proteins of interest.

In some aspects, the disclosure provides cell libraries including cellswith rationally designed edits comprising one or more classes of editsin sequences of interest across the genome of an organism. In specificaspects, the disclosure provides cell libraries including cells withrationally designed edits comprising one or more classes of edits insequences of interest across a subset of the genome. For example, thecell library may include cells with rationally designed edits comprisingone or more classes of edits in sequences of interest across the exome,e.g., every or most open reading frames of the genome. For example, thecell library may include cells with rationally designed edits comprisingone or more classes of edits in sequences of interest across the kinome.In yet another example, the cell library may include cells withrationally designed edits comprising one or more classes of edits insequences of interest across the secretome. In yet other aspects, thecell library may include cells with rationally designed edits created toanalyze various isoforms of proteins encoded within the exome, and thecell libraries can be designed to control expression of one or morespecific isoforms, e.g., for transcriptome analysis.

Importantly, in certain aspects the cell libraries may comprise editsusing randomized sequences, e.g., randomized promoter sequences, toreduce similarity between expression of one or more proteins inindividual cells within the library. Additionally, the promoters in thecell library can be constitutive, inducible or both to enable strongand/or titratable expression.

In other aspects, the present disclosure provides automated editingmethods, automated multi-module cell editing instruments for creating alibrary of cells comprising edits to identify optimum expression of aselected gene target. For example, production of biochemicals throughmetabolic engineering often requires the expression of pathway enzymes,and the best production yields are not always achieved by the highestamount of the target pathway enzymes in the cell, but rather byfine-tuning of the expression levels of the individual enzymes andrelated regulatory proteins and/or pathways. Similarly, expressionlevels of heterologous proteins sometimes can be experimentally adjustedfor optimal yields.

The most obvious way that transcription impacts on gene expressionlevels is through the rate of Pol II initiation, which can be modulatedby combinations of promoter or enhancer strength and trans-activatingfactors (Kadonaga, et al., Cell, 116(2):247-57 (2004)). In eukaryotes,elongation rate may also determine gene expression patterns byinfluencing alternative splicing (Cramer et al., PNAS USA,94(21):11456-60 (1997)). Failed termination on a gene can impair theexpression of downstream genes by reducing the accessibility of thepromoter to Pol II (Greger, et al., PNAS USA, 97(15):8415-20 (2000)).This process, known as transcriptional interference, is particularlyrelevant in lower eukaryotes, as they often have closely spaced genes.

In some embodiments the present disclosure provides methods foroptimizing cellular gene transcription. Gene transcription is the resultof several distinct biological phenomena, including transcriptionalinitiation (RNAp recruitment and transcriptional complex formation),elongation (strand synthesis/extension), and transcriptional termination(RNAp detachment and termination).

Site Directed Mutagenesis

Cell libraries can be created using the automated editing methods,modules, instruments, and systems employing site-directed mutagenesis,i.e., when the amino acid sequence of a protein or other genomic featuremay be altered by deliberately and precisely by mutating the protein orgenomic feature. These cell lines can be useful for various purposes,e.g., for determining protein function within cells, the identificationof enzymatic active sites within cells, and the design of novelproteins. For example, site-directed mutagenesis can be used in amultiplexed fashion to exchange a single amino acid in the sequence of aprotein for another amino acid with different chemical properties. Thisallows one to determine the effect of a rationally designed or randomlygenerated mutation in individual cells within a cell population. See,e.g., Berg, et al. Biochemistry, Sixth Ed. (New York: W.H. Freeman andCompany) (2007).

In another example, edits can be made to individual cells within a celllibrary to substitute amino acids in binding sites, such as substitutionof one or more amino acids in a protein binding site for interactionwithin a protein complex or substitution of one or more amino acids inenzymatic pockets that can accommodate a cofactor or ligand. This classof edits allows the creation of specific manipulations to a protein tomeasure certain properties of one or more proteins, includinginteraction with other cofactors, ligands, etc. within a proteincomplex.

In yet another examples, various edit types can be made to individualcells within a cell library using site specific mutagenesis for studyingexpression quantitative trait loci (eQTLs). An eQTL is a locus thatexplains a fraction of the genetic variance of a gene expressionphenotype. The libraries of the invention would be useful to evaluateand link eQTLs to actual diseased states.

In specific aspects, the edits introduced into the cell libraries of thedisclosure may be created using rational design based on known orpredicted structures of proteins. See, e.g., Chronopoulou and Labrou,Curr Protoc Protein Sci.; Chapter 26: Unit 26.6 (2011). Suchsite-directed mutagenesis can provide individual cells within a librarywith one or more site-directed edits, and preferably two or moresite-directed edits (e.g., combinatorial edits) within a cellpopulation.

In other aspects, cell libraries of the disclosure are created usingsite-directed codon mutation “scanning” of all or substantially all ofthe codons in the coding region of a gene. In this fashion, individualedits of specific codons can be examined for loss-of-function orgain-of-function based on specific polymorphisms in one or more codonsof the gene. These libraries can be a powerful tool for determiningwhich genetic changes are silent or causal of a specific phenotype in acell or cell population. The edits of the codons may be randomlygenerated or may be rationally designed based on known polymorphismsand/or mutations that have been identified in the gene to be analyzed.Moreover, using these techniques on two or more genes in a single in apathway in a cell may determine potential protein:protein interactionsor redundancies in cell functions or pathways.

For example, alanine scanning can be used to determine the contributionof a specific residue to the stability or function of given protein.See, e.g., Lefèvre, et al., Nucleic Acids Research, Volume 25(2):447-448(1997). Alanine is often used in this codon scanning technique becauseof its non-bulky, chemically inert, methyl functional group that canmimic the secondary structure preferences that many of the other aminoacids possess. Codon scanning can also be used to determine whether theside chain of a specific residue plays a significant role in cellfunction and/or activity. Sometimes other amino acids such as valine orleucine can be used in the creation of codon scanning cell libraries ifconservation of the size of mutated residues is needed.

In other specific aspects, cell libraries can be created using theautomated editing methods, automated multi-module cell editinginstruments of the invention to determine the active site of a proteinsuch as an enzyme or hormone, and to elucidate the mechanism of actionof one or more of these proteins in a cell library. Site-directedmutagenesis associated with molecular modeling studies can be used todiscover the active site structure of an enzyme and consequently itsmechanism of action. Analysis of these cell libraries can provide anunderstanding of the role exerted by specific amino acid residues at theactive sites of proteins, in the contacts between subunits of proteincomplexes, on intracellular trafficking and protein stability/half-lifein various genetic backgrounds.

Saturation Mutagenesis

In some aspects, the cell libraries created using the automated editingmethods and automated multi-module cell editing instruments of thedisclosure may be saturation mutagenesis libraries, in which a singlecodon or set of codons is randomized to produce all possible amino acidsat the position of a particular gene or genes of interest. These celllibraries can be particularly useful to generate variants, e.g., fordirected evolution. See, e.g., Chica, et al., Current Opinion inBiotechnology 16 (4): 378-384 (2005); and Shivange, Current Opinion inChemical Biology, 13 (1): 19-25 (2009).

In some aspects, edits comprising different degenerate codons can beused to encode sets of amino acids in the individual cells in thelibraries. Because some amino acids are encoded by more codons thanothers, the exact ratio of amino acids cannot be equal. In certainaspects, more restricted degenerate codons are used. ‘NNK’ and ‘NNS’have the benefit of encoding all 20 amino acids, but still encode a stopcodon 3% of the time. Alternative codons such as ‘NDT’, ‘DBK’ avoid stopcodons entirely, and encode a minimal set of amino acids that stillencompass all the main biophysical types (anionic, cationic, aliphatichydrophobic, aromatic hydrophobic, hydrophilic, small).

In specific aspects, the non-redundant saturation mutagenesis, in whichthe most commonly used codon for a particular organism is used in thesaturation mutagenesis editing process.

Promoter Swaps and Ladders

One mechanism for analyzing and/or optimizing expression of one or moregenes of interest is through the creation of a “promoter swap” celllibrary, in which the cells comprise genetic edits that have specificpromoters linked to one or more genes of interest. Accordingly, the celllibraries created using the methods, automated multi-module cell editinginstruments of the disclosure may be promoter swap cell libraries, whichcan be used, e.g., to increase or decrease expression of a gene ofinterest to optimize a metabolic or genetic pathway. In some aspects,the promoter swap cell library can be used to identify an increase orreduction in the expression of a gene that affects cell vitality orviability, e.g., a gene encoding a protein that impacts on the growthrate or overall health of the cells. In some aspects, the promoter swapcell library can be used to create cells having dependencies and logicbetween the promoters to create synthetic gene networks. In someaspects, the promoter swaps can be used to control cell to cellcommunication between cells of both homogeneous and heterogeneous(complex tissues) populations in nature.

The cell libraries can utilize any given number of promoters that havebeen grouped together based upon exhibition of a range of expressionstrengths and any given number of target genes. The ladder of promotersequences vary expression of at least one locus under at least onecondition. This ladder is then systematically applied to a group ofgenes in the organism using the automated editing methods, automatedmulti-module cell editing instruments of the disclosure.

In specific aspects, the cell library formed using the automated editingprocesses, modules and systems of the disclosure include individualcells that are representative of a given promoter operably linked to oneor more target genes of interest in an otherwise identical geneticbackground. Examples of non-automated editing strategies that can bemodified to utilize the automated systems can be found, e.g., in U.S.Pat. No. 9,988,624.

In specific aspects, the promoter swap cell library is produced byediting a set of target genes to be operably linked to a pre-selectedset of promoters that act as a “promoter ladder” for expression of thegenes of interest. For example, the cells are edited so that one or moreindividual genes of interest are edited to be operably linked with thedifferent promoters in the promoter ladder. When an endogenous promoterdoes not exist, its sequence is unknown, or it has been previouslychanged in some manner, the individual promoters of the promoter laddercan be inserted in front of the genes of interest. These produced celllibraries have individual cells with an individual promoter of theladder operably linked to one or more target genes in an otherwiseidentical genetic context.

The promoters are generally selected to result in variable expressionacross different loci, and may include inducible promoters, constitutivepromoters, or both.

The set of target genes edited using the promoter ladder can include allor most open reading frames (ORFs) in a genome, or a selected subset ofthe genome, e.g., the ORFs of the kinome or a secretome. In someaspects, the target genes can include coding regions for variousisoforms of the genes, and the cell libraries can be designed toexpression of one or more specific isoforms, e.g., for transcriptomeanalysis using various promoters.

The set of target genes can also be genes known or suspected to beinvolved in a particular cellular pathway, e.g. a regulatory pathway orsignaling pathway. The set of target genes can be ORFs related tofunction, by relation to previously demonstrated beneficial edits(previous promoter swaps or previous SNP swaps), by algorithmicselection based on epistatic interactions between previously generatededits, other selection criteria based on hypotheses regarding beneficialORF to target, or through random selection. In specific embodiments, thetarget genes can comprise non-protein coding genes, including non-codingRNAs.

Editing of other functional genetic elements, including insulatorelements and other genomic organization elements, can also be used tosystematically vary the expression level of a set of target genes, andcan be introduced using the methods, automated multi-module cell editinginstruments of the disclosure. In one aspect, a population of cells isedited using a ladder of enhancer sequences, either alone or incombination with selected promoters or a promoter ladder, to create acell library having various edits in these enhancer elements. In anotheraspect, a population of cells is edited using a ladder of ribosomebinding sequences, either alone or in combination with selectedpromoters or a promoter ladder, to create a cell library having variousedits in these ribosome binding sequences.

In another aspect, a population of cells is edited to allow theattachment of various mRNA and/or protein stabilizing or destabilizingsequences to the 5′ or 3′ end, or at any other location, of a transcriptor protein.

In certain aspects, a population of cells of a previously establishedcell line may be edited using the automated editing methods, modules,instruments, and systems of the disclosure to create a cell library toimprove the function, health and/or viability of the cells. For example,many industrial strains currently used for large scale manufacturinghave been developed using random mutagenesis processes iteratively overa period of many years, sometimes decades. Unwanted neutral anddetrimental mutations were introduced into strains along with beneficialchanges, and over time this resulted in strains with deficiencies inoverall robustness and key traits such as growth rates. In anotherexample, mammalian cell lines continue to mutate through the passage ofthe cells over periods of time, and likewise these cell lines can becomeunstable and acquire traits that are undesirable. The automated editingmethods, automated multi-module cell editing instruments of thedisclosure can use editing strategies such as SNP and/or STR swapping,indel creation, or other techniques to remove or change the undesirablegenome sequences and/or introducing new genome sequences to address thedeficiencies while retaining the desirable properties of the cells.

When recursive editing is used, the editing in the individual cells inthe edited cell library can incorporate the inclusion of “landing pads”in an ectopic site in the genome (e.g., a CarT locus) to optimizeexpression, stability and/or control.

In some embodiments, each library produced having individual cellscomprising one or more edits (either introducing or removing) iscultured and analyzed under one or more criteria (e.g., production of achemical or product of interest). The cells possessing the specificcriteria are then associated, or correlated, with one or more particularedits in the cell. In this manner, the effect of a given edit on anynumber of genetic or phenotypic traits of interest can be determined.The identification of multiple edits associated with particular criteriaor enhanced functionality/robustness may lead to cells with highlydesirable characteristics.

Knock-Out or Knock-in Libraries

In certain aspects, the present disclosure provides automated editingmethods, modules, instruments and systems for creating a library ofcells having “knock-out” (KO) or “knock-in” (KI) edits of various genesof interest. Thus, the disclosure is intended to cover edited celllibraries created by the automated editing methods, automatedmulti-module cell editing instruments of the disclosure that have one ormore mutations that remove or reduce the expression of selected genes ofinterest to interrogate the effect of these edits on gene function inindividual cells within the cell library.

The cell libraries can be created using targeted gene KO (e.g., viainsertion/deletion) or KOs (e.g., via homologous directed repair). Forexample, double strand breaks are often repaired via the non-homologousend joining DNA repair pathway. The repair is known to be error prone,and thus insertions and deletions may be introduced that can disruptgene function. Preferably the edits are rationally designed tospecifically affect the genes of interest, and individual cells can becreated having a KI or KI of one or more locus of interest. Cells havinga KO or KI of two or more loci of interest can be created usingautomated recursive editing of the disclosure.

In specific aspects, the KO or KI cell libraries are created usingsimultaneous multiplexed editing of cells within a cell population, andmultiple cells within a cell population are edited in a single round ofediting, i.e., multiple changes within the cells of the cell library arein a single automated operation. In other specific aspects, the celllibraries are created using recursive editing of individual cells withina cell population, and results in the amalgamation of multiple edits oftwo or more sites in the genome into single cells.

SNP or Short Tandem Repeat Swaps

In one aspect, cell libraries are created using the automated editingmethods, automated multi-module cell editing instruments of thedisclosure by systematic introducing or substituting single nucleotidepolymorphisms (“SNPs”) into the genomes of the individual cells tocreate a “SNP swap” cell library. In some embodiments, the SNP swappingmethods of the present disclosure include both the addition ofbeneficial SNPs, and removing detrimental and/or neutral SNPs. The SNPswaps may target coding sequences, non-coding sequences, or both.

In another aspect, a cell library is created using the automated editingmethods, modules, instruments, instruments, and systems of thedisclosure by systematic introducing or substituting short tandemrepeats (“STR”) into the genomes of the individual cells to create an“STR swap” cell library. In some embodiments, the STR swapping methodsof the present disclosure include both the addition of beneficial STRs,and removing detrimental and/or neutral STRs. The STR swaps may targetcoding sequences, non-coding sequences, or both.

In some embodiments, the SNP and/or STR swapping used to create the celllibrary is multiplexed, and multiple cells within a cell population areedited in a single round of editing, i.e., multiple changes within thecells of the cell library are in a single automated operation. In otherembodiments, the SNP and/or STR swapping used to create the cell libraryis recursive, and results in the amalgamation of multiple beneficialsequences and/or the removal of detrimental sequences into single cells.Multiple changes can be either a specific set of defined changes or apartly randomized, combinatorial library of mutations. Removal ofdetrimental mutations and consolidation of beneficial mutations canprovide immediate improvements in various cellular processes. Removal ofgenetic burden or consolidation of beneficial changes into a strain withno genetic burden also provides a new, robust starting point foradditional random mutagenesis that may enable further improvements.

SNP swapping overcomes fundamental limitations of random mutagenesisapproaches as it is not a random approach, but rather the systematicintroduction or removal of individual mutations across cells.

Splice Site Editing

RNA splicing is the process during which introns are excised and exonsare spliced together to create the mRNA that is translated into aprotein. The precise recognition of splicing signals by cellularmachinery is critical to this process. Accordingly, in some aspects, apopulation of cells is edited using a systematic editing to known and/orpredicted splice donor and/or acceptor sites in various loci to create alibrary of splice site variants of various genes. Such editing can helpto elucidate the biological relevance of various isoforms of genes in acellular context. Sequences for rational design of splicing sites ofvarious coding regions, including actual or predicted mutationsassociated with various mammalian disorders, can be predicted usinganalysis techniques such as those found in Nalla and Rogan, Hum Mutat,25:334-342 (2005); Divina, et al., Eur J Hum Genet, 17:759-765 (2009);Desmet, et el., Nucleic Acids Res, 37:e67 (2009); Faber, et al., BMCBioinformatics, 12(suppl 4):S2 (2011).

Start/Stop Codon Exchanges and Incorporation of Nucleic Acid Analogs

In some aspects, the present disclosure provides for the creation ofcell libraries using the automated editing methods, modules, instrumentsand systems of the disclosure, where the libraries are created byswapping start and stop codon variants throughout the genome of anorganism or for a selected subset of coding regions in the genome, e.g.,the kinome or secretome. In the cell library, individual cells will haveone or more start or stop codons replacing the native start or stopcodon for one or more gene of interest.

For example, typical start codons used by eukaryotes are ATG (AUG) andprokaryotes use ATG (AUG) the most, followed by GTG (GUG) and TTG (UUG).The cell library may include individual cells having substitutions forthe native start codons for one or more genes of interest.

In some aspects, the present disclosure provides for automated creationof a cell library by replacing ATG start codons with TTG in front ofselected genes of interest. In other aspects, the present disclosureprovides for automated creation of a cell library by replacing ATG startcodons with GTG. In other aspects, the present disclosure provides forautomated creation of a cell library by replacing GTG start codons withATG. In other aspects, the present disclosure provides for automatedcreation of a cell library by replacing GTG start codons with TTG. Inother aspects, the present disclosure provides for automated creation ofa cell library by replacing TTG start codons with ATG. In other aspects,the present disclosure provides for automated creation of a cell libraryby replacing TTG start codons with GTG.

In other examples, typical stop codons for S. cerevisiae and mammals areTAA (UAA) and TGA (UGA), respectively. The typical stop codon formonocotyledonous plants is TGA (UGA), whereas insects and E. colicommonly use TAA (UAA) as the stop codon (Dalphin, et al., Nucl. AcidsRes., 24: 216-218 (1996)). The cell library may include individual cellshaving substitutions for the native stop codons for one or more genes ofinterest.

In some aspects, the present disclosure provides for automated creationof a cell library by replacing TAA stop codons with TAG. In otheraspects, the present disclosure provides for automated creation of acell library by replacing TAA stop codons with TGA. In other aspects,the present disclosure provides for automated creation of a cell libraryby replacing TGA stop codons with TAA. In other aspects, the presentdisclosure provides for automated creation of a cell library byreplacing TGA stop codons with TAG. In other aspects, the presentdisclosure provides for automated creation of a cell library byreplacing TAG stop codons with TAA. In other aspects, the presentinvention teaches automated creation of a cell library by replacing TAGstop codons with TGA.

Terminator Swaps and Ladders

One mechanism for identifying optimum termination of a pre-spliced mRNAof one or more genes of interest is through the creation of a“terminator swap” cell library, in which the cells comprise geneticedits that have specific terminator sequences linked to one or moregenes of interest. Accordingly, the cell libraries created using themethods, modules, instruments and systems of the disclosure may beterminator swap cell libraries, which can be used, e.g., to affect mRNAstability by releasing transcripts from sites of synthesis. In otherembodiments, the terminator swap cell library can be used to identify anincrease or reduction in the efficiency of transcriptional terminationand thus accumulation of unspliced pre-mRNA (e.g., West and Proudfoot,Mol Cell.; 33(3-9); 354-364 (2009)) and/or 3′ end processing (e.g.,West, et al., Mol Cell. 29(5):600-10 (2008)). In the case where a geneis linked to multiple termination sites, the edits may edit acombination of edits to multiple terminators that are associated with agene. Additional amino acids may also be added to the ends of proteinsto determine the effect on the protein length on terminators.

The cell libraries can utilize any given number of edits of terminatorsthat have been selected for the terminator ladder based upon exhibitionof a range of activity and any given number of target genes. The ladderof terminator sequences vary expression of at least one locus under atleast one condition. This ladder is then systematically applied to agroup of genes in the organism using the automated editing methods,modules, instruments and systems of the disclosure.

In some aspects, the present disclosure provides for the creation ofcell libraries using the automated editing methods, modules, instrumentsand systems of disclosure, where the libraries are created to editterminator signals in one or more regions in the genome in theindividual cells of the library. Transcriptional termination ineukaryotes operates through terminator signals that are recognized byprotein factors associated with the RNA polymerase II. For example, thecell library may contain individual eukaryotic cells with edits in genesencoding polyadenylation specificity factor (CPSF) and cleavagestimulation factor (CstF) and or gene encoding proteins recruited byCPSF and CstF factors to termination sites. In prokaryotes, twoprincipal mechanisms, termed Rho-independent and Rho-dependenttermination, mediate transcriptional termination. For example, the celllibrary may contain individual prokaryotic cells with edits in genesencoding proteins that affect the binding, efficiency and/or activity ofthese termination pathways.

In certain aspects, the present disclosure provides methods of selectingtermination sequences (“terminators”) with optimal properties. Forexample, in some embodiments, the present disclosure teaches providesmethods for introducing and/or editing one or more terminators and/orgenerating variants of one or more terminators within a host cell, whichexhibit a range of activity. A particular combination of terminators canbe grouped together as a terminator ladder, and cell libraries of thedisclosure include individual cells that are representative ofterminators operably linked to one or more target genes of interest inan otherwise identical genetic background. Examples of non-automatedediting strategies that can be modified to utilize the automatedinstruments can be found, e.g., in U.S. Pat. No. 9,988,624 to Serber etal., entitled “Microbial strain improvement by a HTP genomic engineeringplatform.”

In specific aspects, the terminator swap cell library is produced byediting a set of target genes to be operably linked to a pre-selectedset of terminators that act as a “terminator ladder” for expression ofthe genes of interest. For example, the cells are edited so that theendogenous promoter is operably linked to the individual genes ofinterest are edited with the different promoters in the promoter ladder.When the endogenous promoter does not exist, its sequence is unknown, orit has been previously changed in some manner, the individual promotersof the promoter ladder can be inserted in front of the genes ofinterest. These produced cell libraries have individual cells with anindividual promoter of the ladder operably linked to one or more targetgenes in an otherwise identical genetic context. The terminator ladderin question is then associated with a given gene of interest.

The terminator ladder can be used to more generally affect terminationof all or most ORFs in a genome, or a selected subset of the genome,e.g., the ORFs of a kinome or a secretome. The set of target genes canalso be genes known or suspected to be involved in a particular cellularpathway, e.g. a regulatory pathway or signaling pathway. The set oftarget genes can be ORFs related to function, by relation to previouslydemonstrated beneficial edits (previous promoter swaps or previous SNPswaps), by algorithmic selection based on epistatic interactions betweenpreviously generated edits, other selection criteria based on hypothesesregarding beneficial ORF to target, or through random selection. Inspecific embodiments, the target genes can comprise non-protein codinggenes, including non-coding RNAs.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Example 1: Production and Transformation of Electrocompetent E. coli

For testing transformation of the FTEP device, such as the FTEP deviceconfigured as shown in FIGS. 10B-10D (vi), electrocompetent E. colicells were created. To create a starter culture, 6 ml volumes of LBchlor-25 (LB with 25 μg/ml chloramphenicol) were transferred to 14 mlculture tubes. A 25 μl aliquot of E. coli was used to inoculate the LBchlor-25 tubes. Following inoculation, the tubes were placed at a 45°angle in the shaking incubator set to 250 RPM and 30° C. for overnightgrowth, between 12-16 hrs. The OD600 value should be between 2.0 and4.0. A 1:100 inoculum volume of the 250 ml LB chlor-25 tubes weretransferred to four sterile 500 ml baffled shake flasks, i.e., 2.5 mlper 250 ml volume shake flask. The flasks were placed in a shakingincubator set to 250 RPM and 30° C. The growth was monitored bymeasuring OD600 every 1 to 2 hr. When the OD600 of the culture wasbetween 0.5-0.6 (approx. 3-4 hrs), the flasks were removed from theincubator. The cells were centrifuged at 4300 RPM, 10 min, 4° C. Thesupernatant was removed, and 100 ml of ice-cold 10% glycerol wastransferred to each sample. The cells were gently resuspended, and thewash procedure performed three times, each time with the cellsresuspended in 10% glycerol. After the fourth centrifugation, the cellresuspension was transferred to a 50 ml conical Falcon tube andadditional ice-cold 10% glycerol added to bring the volume up to 30 ml.The cells were again centrifuged at 4300 RPM, 10 min, 4° C., thesupernatant removed, and the cell pellet resuspended in 10 ml ice-coldglycerol. The cells are aliquoted in 1:100 dilutions of cell suspensionand ice-cold glycerol.

The comparative electroporation experiment was performed to determinethe efficiency of transformation of the electrocompetent E. coli usingthe embodiment of the FTEP device shown at (ii), (iii), and (vi) ofFIGS. 10B and 10C and (ii) and (vi) of FIG. 10D. The flow rate wascontrolled with a pressure control system. The suspension of cells withDNA was loaded into the FTEP inlet reservoir. The transformed cellsflowed directly from the inlet and inlet channel, through the flowchannel, through the outlet channel, and into the outlet containingrecovery medium. The cells were transferred into a tube containingadditional recovery medium, placed in an incubator shaker at 30° C.shaking at 250 rpm for 3 hours. The cells were plated to determine thecolony forming units (CFUs) that survived electroporation and failed totake up a plasmid and the CFUs that survived electroporation and took upa plasmid. Plates were incubated at 30° C.; E. coli colonies werecounted after 24 hrs.

The flow-through electroporation experiments were benchmarked against 2mm electroporation cuvettes (Bulldog Bio, Portsmouth, N.H.) using an invitro high voltage electroporator (NEPAGENE™ ELEPO21). Stock tubes ofcell suspensions with DNA were prepared and used for side-to-sideexperiments with the NEPAGENE™ and the flow-through electroporation. Theresults are shown in FIG. 19A. In FIG. 19A, the left-most bars hatched/// denote cell input, the bars to the left bars hatched \\\ denote thenumber of cells that survived transformation, and the right bars hatched/// denote the number of cells that were actually transformed. The FTEPdevice showed equivalent transformation of electrocompetent E. colicells at various voltages as compared to the NEPAGENE™ electroporator.As can be seen, the transformation survival rate is at least 90% and insome embodiments is at least 95%, 96%, 97%, 98%, or 99%. The recoveryratio (the fraction of introduced cells which are successfullytransformed and recovered) is in certain embodiments at least 0.001 andpreferably between 0.00001 and 0.01. In FIG. 19A the recovery ratio isapproximately 0.0001.

Additionally, a comparison of the NEPAGENE™ ELEPO21 and the FTEP devicewas made for efficiencies of transformation (uptake), cutting, andediting. In FIG. 19B, triplicate experiments were performed where thebars hatched /// denote the number of cells input for transformation,and the bars hatched \\\ denote the number of cells that weretransformed (uptake), the number of cells where the genome of the cellswas cut by a nuclease transcribed and translated from a vectortransformed into the cells (cutting), and the number of cells whereediting was effected (cutting and repair using a nuclease transcribedand translated from a vector transformed into the cells, and using aguide RNA and a donor DNA sequence both of which were transcribed from avector transformed into the cells). In addition, note that innon-editing cell lines, the number of colonies for both the NEPAGENE™electroporator and the FTEP showed equivalent transformationefficiencies. Moreover, it can be seen that the FTEP showed equivalenttransformation, cutting, and editing efficiencies as the NEPAGENE™electroporator.

Example 2: Production and Transformation of Electrocompetent S.Cerevisiae

For further testing transformation of the FTEP device, such as the FTEPdevice configured as shown in FIGS. 10B-10D (vi), S. Cerevisiae cellswere prepared using the methods as generally set forth in Bergkessel andGuthrie, Methods Enzymol., 529:311-20 (2013). Briefly, YFAP media wasinoculated for overnight growth, with 3 ml inoculate to produce 100 mlof cells. Every 100 ml of culture processed resulted in approximately 1ml of competent cells. Cells were incubated at 30° C. in a shakingincubator until they reached an OD600 of 1.5+/−0.1.

A conditioning buffer was prepared using 100 mM lithium acetate, 10 mMdithiothreitol, and 50 mL of buffer for every 100 mL of cells grown andkept at room temperature. Cells were harvested in 250 ml bottles at 4300rpm for 3 minutes, and the supernatant removed. The cell pellets weresuspended in 100 ml of cold 1 M sorbitol, spun at 4300 rpm for 3 minutesand the supernatant once again removed. The cells were suspended inconditioning buffer, then the suspension transferred into an appropriateflask and shaken at 200 RPM and 30° C. for 30 minutes. The suspensionswere transferred to 50 ml conical vials and spun at 4300 rpm for 3minutes. The supernatant was removed and the pellet resuspended in cold1 M sorbitol. These steps were repeated three times for a total of threewash-spin-decant steps. The pellet was suspended in sorbitol to a finalOD of 150+/−20.

A comparative electroporation experiment was performed to determine theefficiency of transformation of the electrocompetent S. Cerevisiae usingthe FTEP device. The flow rate was controlled with a syringe pump(Harvard apparatus PHD ULTRA™ 4400). The suspension of cells with DNAwas loaded into a 1 mL glass syringe (Hamilton 81320 Syringe, PTFE LuerLock) before mounting on the pump. The output from the functiongenerator was turned on immediately after starting the flow. Theprocessed cells flowed directly into a tube with 1M sorbitol withcarbenicillin. Cells were collected until the same volume electroporatedin the NEPAGENE™ had been processed, at which point the flow and theoutput from the function generator were stopped. After a 3-hour recoveryin an incubator shaker at 30° C. and 250 rpm, cells were plated todetermine the colony forming units (CFUs) that survived electroporationand failed to take up a plasmid and the CFUs that survivedelectroporation and took up a plasmid. Plates were incubated at 30° C.Yeast colonies are counted after 48-76 hrs.

The flow-through electroporation experiments were benchmarked against 2mm electroporation cuvettes (Bulldog Bio, Portsmouth, N.H.) using an invitro high voltage electroporator (NEPAGENE™ ELEPO21). Stock tubes ofcell suspensions with DNA were prepared and used for side-to-sideexperiments with the NEPAGENE™ and the flow-through electroporation. Theresults are shown in FIG. 20. The device showed better transformationand survival of electrocompetent S. Cerevisiae at 2.5 kV voltages ascompared to the NEPAGENE™ method. Input is total number of cells thatwere processed.

Example 3: FTEP Pressure Sensing and Flow Rates

The pressure and sensing was also tested using an FTEP devicesubstantially as shown in FIGS. 1-B-10D (vi) as part of a cartridgedevice as illustrated in FIG. 1A. An inline flow sensor measurement wasused to indicate when, after the liquid containing the cells and DNAflowed through the FTEP chip, where the inlet reservoir was emptied.Approximately 65 μL of liquid was loaded into the input reservoir andthe automated FTEP module was powered on. Looking at the graph at thetop of FIG. 21, it can be seen that after a few short startuptransients, the flow rate shows about—3 standard cubic centimeters perminute (SCCM) of flow for almost 8 seconds (8000 ms) until it jumps to24 SCCM. This transition occurs at an end of run trigger, which is anindicator that the liquid containing the cells and DNA has beenprocessed through the FTEP device and that air is not flowing throughthe FTEP device. That trigger may constitute detection of an increaseflow rate or a sudden fluctuation (increase or decrease) in the pressureof the air (such as at a conduit leading from a syringe pump). In onepreferred embodiment, the flow sensor in FIG. 27 detects an increase inair flow indicative of the fluid being completely drained from the inputreservoir. At this point, pressure may be reversed to allow a multi-passelectroporation procedure; that is, cells to electroporated may be“pulled” from the inlet toward the outlet for one pass ofelectroporation, and once the inlet reservoir is emptied, the sensor mayreverse the pressure where the liquid and cells/DNA is “pushed” from theoutlet end of the flow-through FTEP device toward the inlet end to passbetween the electrodes again for another pass of electroporation. Thisprocess may be repeated one to many times. Alternatively, the pressuremay be stopped entirely and the transformed cells in the outletretrieved.

The multi-cycle approach may be particularly advantageous in that itlimits the dwell time of the cells and exogenous materials in theelectric filed which may in turn prevent cell damage and increasesurvival rates. The back-and-forth process may be repeated 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 times. FIG. 27 at bottom shows a simple depictionof the pressure system and FTEP. The pressure manifold is mated to theupwardly-extending reservoirs via one or more complementary seals orgaskets disposed on the manifold or the reservoirs.

Example 4: Fully-Automated Singleplex RGN-directed Editing Run

Singleplex automated genomic editing using MAD7 nuclease wassuccessfully performed with an automated multi-module instrument of thedisclosure. See U.S. Pat. No. 9,982,279.

An ampR plasmid backbone and a lacZ_F172* editing cassette wereassembled via Gibson Assembly® into an “editing vector” in an isothermalnucleic acid assembly module included in the automated instrument.lacZ_F172 functionally knocks out the lacZ gene. “lacZ_F172*” indicatesthat the edit happens at the 172nd residue in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The assembled editing vector andrecombineering-ready, electrocompetent E. Coli cells were transferredinto a transformation module for electroporation. The transformationmodule comprised an ADP-EPC cuvette. See, e.g., U.S. Pat No. 62/551,069.The cells and nucleic acids were combined and allowed to mix for 1minute, and electroporation was performed for 30 seconds. The parametersfor the poring pulse were: voltage, 2400 V; length, 5 ms; interval, 50ms; number of pulses, 1; polarity, +. The paramters for the transferpulses were: Voltage, 150 V; length, 50 ms; interval, 50 ms; number ofpulses, 20; polarity, +/−. Following electroporation, the cells weretransferred to a recovery module (another growth module), and allowed torecover in SOC medium containing chloramphenicol. Carbenicillin wasadded to the medium after 1 hour, and the cells were allowed to recoverfor another 2 hours. After recovery, the cells were held at 4° C. untilrecovered by the user.

After the automated process and recovery, an aliquot of cells was platedon a petri dish containing a MacConkey agar base supplemented withlactose (as the sugar substrate), chloramphenicol and carbenicillin andthe inoculates grown until visible colonies appeared. White coloniesrepresented functionally edited cells, purple colonies representedun-edited cells. All liquid transfers were performed by the automatedliquid handling device of the automated multi-module cell editinginstrument.

The result of the automated processing was that approximately 1.0E⁻⁰³total cells were transformed (comparable to conventional benchtopresults), and the editing efficiency was 83.5%. The lacZ_172 edit in thewhite colonies was confirmed by sequencing of the edited region of thegenome of the cells. Further, steps of the automated cell processingwere observed remotely by webcam and text messages were sent to updatethe status of the automated processing procedure.

Example 5: Fully-Automated Recursive Editing Run

Recursive editing was successfully achieved using the automatedmulti-module cell editing instrument. An ampR plasmid backbone and alacZ_V10* editing cassette were assembled via Gibson Assembly® into an“editing vector” in an isothermal nucleic acid assembly module includedin the automated system. Similar to the lacZ_F172 edit, the lacZ_V10edit functionally knocks out the lacZ gene. “lacZ_V10” indicates thatthe edit happens at amino acid position 10 in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The first assembled editing vectorand the recombineering-ready electrocompetent E. Coli cells weretransferred into a transformation module, such as FTEP devicesubstantially as shown in FIGS. 1-B-10D (vi) as part of a cartridgedevice as illustrated in FIG. 1A, for electroporation. Thetransformation module comprised an ADP-EPC cuvette. The cells andnucleic acids were combined and allowed to mix for 1 minute, andelectroporation was performed for 30 seconds. The parameters for theporing pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms;number of pulses, 1; polarity, +. The parameters for the transfer pulseswere: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses,20; polarity, +/−. Following electroporation, the cells were transferredto a recovery module (another growth module) allowed to recover in SOCmedium containing chloramphenicol. Carbenicillin was added to the mediumafter 1 hour, and the cells were grown for another 2 hours. The cellswere then transferred to a centrifuge module and a media exchange wasthen performed. Cells were resuspended in TB containing chloramphenicoland carbenicillin where the cells were grown to OD600 of 2.7, thenconcentrated and rendered electrocompetent.

During cell growth, a second editing vector was prepared in theisothermal nucleic acid assembly module. The second editing vectorcomprised a kanamycin resistance gene, and the editing cassettecomprised a galK Y145* edit. If successful, the galK Y145* edit conferson the cells the ability to uptake and metabolize galactose. The editgenerated by the galK Y154* cassette introduces a stop codon at the154th amino acid reside, changing the tyrosine amino acid to a stopcodon. This edit makes the galK gene product non-functional and inhibitsthe cells from being able to metabolize galactose. Following assembly,the second editing vector product was de-salted in the isothermalnucleic acid assembly module using AMPure beads, washed with 80%ethanol, and eluted in buffer. The assembled second editing vector andthe electrocompetent E. Coli cells (that were transformed with andselected for the first editing vector) were transferred into atransformation module, FTEP device substantially as shown in FIGS.1-B-10D (vi) as part of a cartridge device as illustrated in FIG. 1A,for electroporation, using the same parameters as detailed above.Following electroporation, the cells were transferred to a recoverymodule (another growth module), allowed to recover in SOC mediumcontaining carbenicillin. After recovery, the cells were held at 4° C.until retrieved, after which an aliquot of cells were plated on LB agarsupplemented with chloramphenicol, and kanamycin. To quantify both lacZand galK edits, replica patch plates were generated on two mediatypes: 1) MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol, and kanamycin, and 2) MacConkey agar basesupplemented with galactose (as the sugar substrate), chloramphenicol,and kanamycin. All liquid transfers were performed by the automatedliquid handling device of the automated multi-module cell editinginstrument.

In this recursive editing experiment, 41% of the colonies screened hadboth the lacZ and galK edits, the results of which were comparable tothe double editing efficiencies obtained using a “benchtop” or manualapproach.

While certain embodiments have been described, these embodiments havebeen presented by way of example only, and are not intended to limit thescope of the present disclosures. Indeed, the novel methods,apparatuses, modules, instruments and systems described herein can beembodied in a variety of other forms; furthermore, various omissions,substitutions and changes in the form of the methods, apparatuses,modules, instruments and systems described herein can be made withoutdeparting from the spirit of the present disclosures. The accompanyingclaims and their equivalents are intended to cover such forms ormodifications as would fall within the scope and spirit of the presentdisclosures.

The invention claimed is:
 1. An automated multi-module cell editinginstrument comprising: a housing configured to house all of some of themodules; one or more receptacles configured to receive cells and nucleicacids; a flow-through electroporation (FTEP) module configured tointroduce the nucleic acids into the cells, wherein the FTEP modulecomprises: a. at least a first inlet and a first inlet channel forintroducing a fluid comprising the cells and the nucleic acids into theFTEP module; b. an outlet and an outlet channel for removing a fluidcomprising transformed cells from the FTEP module; c. a flow channelintersecting and positioned between a first inlet channel and the outletchannel; and d. two or more electrodes positioned in the flow channelbetween the intersection of the flow channel with the first inletchannel and the intersection of the flow channel with the outletchannel, wherein the electrodes apply one or more electric pulses to thecells in the fluid as they pass through the flow channel therebyintroducing the nucleic acids into the cells in the fluid; a selectionmodule configured to select for transformed cells; an editing moduleconfigured to allow the nucleic acids to edit nucleic acids in thetransformed cells; a processor configured to operate the automatedmulti-module cell editing instrument based on user input and/orselection of a pre-programmed script; and an automated liquid handlingsystem to move liquids from the one or more receptacles to the FTEPmodule, from the FTEP module to the selection module, and from theselection module to the editing module, all without user intervention.2. The automated multi-module cell editing instrument of claim 1 whereinthe selection module is also the editing module.
 3. The automatedmulti-module cell editing instrument of claim 1 wherein the selectionmodule is separate from the editing module.
 4. The automatedmulti-module cell editing instrument of claim 1 wherein the FTEP modulefurther comprises a second inlet and a second inlet channel.
 5. Theautomated multi-module cell editing instrument of claim 4 wherein thesecond inlet and second inlet channel of the FTEP module are locatedbetween the first inlet and first inlet channel and the electrodes ofthe FTEP module.
 6. The automated multi-module cell editing instrumentof claim 4 wherein the second inlet and second inlet channel of the FTEPmodule are located between the electrodes and the outlet channel andoutlet of the FTEP module.
 7. The automated multi-module cell editinginstrument of claim 1 wherein the flow channel is constricted betweenthe first inlet and first inlet channel and the outlet and outletchannel.
 8. The automated multi-module cell editing instrument of claim1 wherein the FTEP module further comprises a filter between the firstinlet channel and the electrodes.
 9. The automated multi-module cellediting instrument of claim 1, wherein device is configured for use withbacterial, yeast and mammalian cells.
 10. The automated multi-modulecell editing instrument of claim 1, further comprising a reagentcartridge.
 11. The automated multi-module cell editing instrument ofclaim 10, wherein the FTEP is part of the reagent cartridge.
 12. Anautomated multi-module cell editing instrument comprising a flow-throughelectroporation (FTEP) module, wherein the multi-module cell editinginstrument comprises: a housing configured to house all of some of themodules; one or more receptacles configured to receive nucleic acids andcells; a growth module in which to grow the cells; the FTEP moduleconfigured to introduce the nucleic acids into the cells; wherein theFTEP module comprises: a. at least a first inlet and at least a firstinlet channel for introducing a fluid comprising cells and nucleic acidsto the FTEP module; b. an outlet and an outlet channel for removingtransformed cells and exogenous material from the FTEP module; c. a flowchannel positioned between the first inlet channel and the outletchannel; and d. an electrode positioned on either side of the flowchannel, wherein the electrodes apply one or more electric pulses to thecells in the fluid as they pass through the flow channel, therebyintroducing the nucleic acids into the cells in the fluid; a selectionmodule; an editing module configured to allow the introduced nucleicacids to edit nucleic acids in the cells; a processor configured tooperate the automated multi-module cell editing instrument based on userinput and/or selection of a pre-programmed script; and an automatedliquid handling system to move liquids from the one or more receptaclesto the growth module, from the growth module to the FTEP module, fromthe FTEP module to the selection module, from the selection module tothe editing module, all without user intervention.
 13. The automatedmulti-module cell editing instrument of claim 12 wherein the selectionmodule comprises an antibiotic.
 14. The automated multi-module cellediting instrument of claim 12 wherein the selection module is separatefrom the editing module.
 15. The automated multi-module cell editinginstrument of claim 12 wherein the selection module is combined with theediting module.
 16. The automated multi-module cell editing instrumentof claim 12 wherein the electrodes define a narrowed portion of the flowchannel.
 17. The automated multi-module cell editing instrument of claim16 wherein the electrodes are in direct contact with the fluid in theflow channel.
 18. The automated multi-module cell editing instrument ofclaim 12 wherein the FTEP module further comprises a filter between theat least first inlet channel and the electrodes.
 19. The automatedmulti-module cell editing instrument of claim 12 further comprising areagent cartridge.
 20. The automated multi-module cell editinginstrument of claim 19 wherein the FTEP is a component of the reagentcartridge.